[1,2,4]triazolo[4,3-b]pyridazines for use in the treatment of proliferative diseases

ABSTRACT

The invention concerns compounds of Formula (I) (Formula (I)) or pharmaceutically-acceptable salts thereof, wherein R 1 , R 2  and n have any of the meanings defined herein before in the description; processes for their preparation, pharmaceutical compositions containing them and their use as anti-proliferative and/or cell-killing agents.

The invention concerns certain substituted triazolopyridazine (TPDZ)compounds or pharmaceutically acceptable salts thereof, which possessanti-cancer activity and are accordingly useful in methods of treatmentof the human or animal body. The invention also concerns a process forthe manufacture of said TPDZ compounds, pharmaceutical compositionscomprising said compounds or pharmaceutically acceptable salts thereof,and to methods of treatment of cancers in warm-blooded animals such asman.

The invention also relates to TPDZ compounds that are inhibitors of oneor more bromodomain-containing proteins, in particular the BET family ofbromodomain-containing proteins.

Bromodomain-containing proteins are implicated in diverse diseases andare of substantial interest as therapeutic targets. The bromodomain is ahighly conserved structural fold that recognizes acetylated-lysineresidues and is found in large multidomain proteins associated withchromatin remodeling transcription control, methyl or acetyltransferaseactivity or helicases. The BET family of bromodomain containing proteinsis comprised of four members (BRD2, BRD3, BRD4, and BRDt) which alldisplay a common domain architecture of N-terminal tandem bromodomainscapable of binding to acetylated lysine residues in histones andtranscription factors. BRD4 plays an important role in genetranscriptional regulation as evidenced by its association with thepositive transcription elongation factor b (pTEFb) (Jang et al. Mol.Cell, 2005, 19, 523-534), general transcription cofactor Mediator(Chiang, F 1000 Biol. Rep, 2009, 1, 98), gene-specific pro-inflammatoryfactor NFkB (Huang et al. Mol. Cell Biol. 2009, 29, 1375-1387) andvirus-encoded transcriptional regulators (You et al. Cell, 2004, 117,349-360). It is observed that BRD4 has asymmetrical loading at extralarge enhancers that are associated with a small subset of genes whichoften constitute the oncogenic and lineage-specific transcriptionalprograms in a particular cellular context (Loven et al. Cell, 2013, 153,320-334). Similarly, BRD2 and BRD3 are reported as transcriptionregulators binding to hyper-acetylated chromatin regions of growthpromoting genes (LeRoy et al. Mol. Cell. 2008, 30, 51-60). It has alsobeen reported that BRD4 or BRD3 may fuse with NUT (nuclear protein intestis) forming novel oncogenes, BRD4-NUT or BRD3-NUT, in a highlymalignant form of epithelial neoplasia (French et al. Cancer Research,2003, 63, 304-307 and French et al. Journal Clinical Oncology, 2004, 22,4135-4139). Data suggests that BRD-NUT fusion proteins contribute tocarcinogenesis (French et al. 2008, Oncogene, 27, 2237-2242).). It isalso found that BRD4 gene is altered in the form of gene amplificationin serous ovarian and other cancers in The Cancer Genome Atlas (TCGA)dataset. All BET family members have been reported to have some functioncontrolling or executing aspects of the cell cycle and have been shownto remain in complex with chromosomes during cell division, suggesting arole in the maintenance of epigenetic memory. Not surprisingly, BETfamily members were recently established as being important for themaintenance of many tumor types for example, acute myeloid and mixedlineage leukemia (AML), multiple myeloma (MM), lymphoma, glioblastomaand neuroblastoma. BRD4 inhibition potently suppresses Myc, ER, BC12,and other oncogenes which are frequently altered in cancer. Modulationof these key genes is believed to contribute to the anti-tumor phenotypeof BET inhibition

In addition, BET inhibitors have been shown to have anti-inflammatoryproperties (Nicodeme et al. Nature, 2010, 468, 1119-1123) and reactivatelatent HIV transcription in cell line models of latency (Banerjee et al.J Leukoc Biol, 2012, 92, 1147-1154).

Recently, a few compounds have been reported as bromodomain inhibitors,for example benzodiazepines derivatives such as those disclosed inWO2011/054553. However, there remains a need for developing novel andpotent bromodomain inhibitors that can be used to treat diseases andindications where bromodomain containing proteins are implicated.

The compounds of the invention have been found to possess activity asinhibitors of bromodomain-containing proteins, such as the BET family ofbromodomains, for example BRD4, BRD2, BRD3 and BRDt, and the tandemdomains thereof, for example BRD4(1) and BRD4(2).

According to one aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof

wherein:—R¹ is the group

or the group

and

denotes the point of attachment;R² is a C₁-C₄alkyl; andn is 2 or 3.

In another aspect of the invention, there is provided a compound ofFormula (I) as defined above.

In a further aspect of the invention R² is methyl.

In yet a further aspect of the invention, R¹ is the group

R² is C₁-C₄ alkyl; and

n is 2.

In one aspect of the invention, the compound of Formula (I) is acompound selected from:

-   4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one;-   1-(4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-3,5-dimethylpiperazin-1-yl)ethanone;-   4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-1,3-dimethylpiperazin-2-one;    and-   1-(4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-3,5-dimethylpiperazin-1-yl)ethanone.

In another aspect of the invention, the compound of Formula (I) is acompound of Formula (IA):

The compound of Formula (IA) is also referred to hereinafter as CompoundA.

In another aspect, the compound of Formula (I) is a compound of Formula(IB):

According to a further aspect of the invention, the compound of Formula(I) is a compound of Formula (IC):

According to a further aspect of the invention, the compound of Formula(I) is a compound of Formula (ID):

A further aspect provides any of the aspects defined herein (for examplethe aspect of claim 1) with the proviso that one or more specificExamples (for instance one, two or three specific Examples) selectedfrom the group consisting of Examples 1, 2, 3 and, 4, is individuallydisclaimed.

Some of the compounds of Formula (I) may be crystalline and may havemore than one crystalline form. It is to be understood that theinvention encompasses any crystalline or amorphous form, or mixturesthereof, which form possess properties useful in BET inhibitory activityand, such as, BRD2, BRD3, BRD4, and BRDt inhibitory activity. It is wellknown how to determine the efficacy of a crystalline or amorphous formby the standard tests described hereinafter.

It is generally known that crystalline materials may be analysed usingconventional techniques such as, for example, X-ray powder diffraction(hereinafter XRPD) analysis and Differential Scanning Calorimetry(hereinafter DSC).

As an example, the compound of Example 1 exhibits crystallinity and onecrystalline form, Form A, has been identified.

Accordingly, a further aspect of the invention is Form A of Compound A(Example 1).

According to the invention, there is provided a crystalline form, FormA, of Compound A which has a XRPD pattern with at least one specificpeak at about 2-theta=20.90, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern with at least one specific peakat about 2-theta=16.7°, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern with at least two specific peaksat about 2-theta=20.90 and 16.70, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern with specific peaks at about2-theta=20.9, 16.7, 20.2, 21.2, 27.4, 18.0, 16.8, 23.6, 15.1 and 15.50,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern substantially the same as theXRPD shown in Figure A, measured using CuKα radiation.

According to a further aspect of the invention, there is provided acrystalline form, Form A, of Compound A which has a XRPD pattern with atleast one specific peak at 2-theta=20.90 plus or minus 0.2° 2-theta,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern with at least one specific peakat 2-theta=16.7° plus or minus 0.2° 2-theta, measured using CuKαradiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a XRPD pattern with at least two specific peaksat 2-theta=20.90 and 16.70 plus or minus 0.2° 2-theta, measured usingCuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A which has a X-ray powder diffraction pattern with specificpeaks at 2-theta=20.9, 16.7, 20.2, 21.2, 27.4, 18.0, 16.8, 23.6, 15.1and 15.50 plus or minus 0.2° 2-theta, measured using CuKα radiation.

When it is stated that the invention relates to a crystalline form ofCompound A, Form A, the degree of crystallinity is conveniently greaterthan about 60%, more conveniently greater than about 80%, preferablygreater than about 90% and more preferably greater than about 95%. Mostpreferably the degree of crystallinity is greater than about 98%.

Some of the compounds of Formula (I) may form co-crystals with specificco-former molecules. It is to be understood that the present inventionencompasses any such co-crystals, which possess properties useful in BETinhibitory activity and, such as, BRD2, BRD3, BRD4, and BRDt inhibitoryactivity It is well known how to determine the efficacy of suchco-crystals by the standard tests described hereinafter.

Accordingly, the invention provides a co-crystal of a compound ofFormula (I) and a co-former molecule.

Accordingly, to a further aspect of the invention there is provided aco-crystal of Compound A and the co-former molecule6-hydroxy-2-naphthoic acid.

For the avoidance of doubt, the term “co-crystal” refers to amulticomponent system in which there exists a host API (activepharmaceutical ingredient) molecule or molecules and a guest (orco-former) molecule or molecules in the same crystal lattice. In aco-crystal, both the API molecule and the guest (or co-former) moleculeexist as solids at room temperature when alone in their pure form (inorder to distinguish the co-crystal from solvates or hydrates). In aco-crystal the API and co-former molecules interact by hydrogen bondingand possibly other non-covalent interactions.

In preparing co-crystals of Compound A with 6-hydroxy-2-naphthoic acid,where Compound A is the API, a range of API:co-former molarratios/stoichiometries may be achieved, for example an overallAPI:co-former molar ratio of 1:1, although this may vary slightly,depending, for example, on the characterisation measurements.Accordingly, the invention provides a co-crystal of Compound A andco-former molecule 6-hydroxy-2-naphthoic acid with a molar ratio ofCompound A:6-hydroxy-2-naphthoic acid in the range 1:0.8 to 1:1.2. Inone aspect of the invention there is provided a CompoundA:6-hydroxy-2-naphthoic acid (1:1) co-crystal.

In a further aspect of the invention the co-crystal of Compound A with6-hydroxy-2-naphthoic acid is in a crystalline form, Form A.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at about 2-theta=19.40,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal, which has aXRPD pattern with at least one specific peak at about 2-theta=12.5°,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least two specific peaks at about 2-theta=19.40 and12.50, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at about 2-theta=19.4, 12.5, 12.8,18.1, 24.2, 23.4, 14.0, 18.6, 17.0, and 17.9°, measured using CuKαradiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern substantially the same as the XRPD pattern shown in FigureI, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at 2-theta=19.40 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern, with at least one specific peak at 2-theta=12.50 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the present invention there is provided a crystalline form,Form A, of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal whichhas a XRPD pattern, with at least two specific peaks at 2-theta=19.40and 12.50 wherein said values may be plus or minus 0.2° 2-theta,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form A,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at 2-theta=19.4, 12.5, 12.8, 18.1,24.2, 23.4, 14.0, 18.6, 17.0 and 17.90 wherein said values may be plusor minus 0.2° 2-theta, measured using CuKα radiation.

In a further aspect of the invention the co-crystal of Compound A with6-hydroxy-2-naphthoic acid is in a crystalline form, Form B. Accordingto the invention there is provided a crystalline form, Form B, ofCompound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at about 2-theta=15.20measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at about 2-theta=6.10,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least two specific peaks at about 2-theta=15.2 and6.10 measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at about 2-theta=15.2, 6.1, 16.8, 12.2,26.1, 28.4, 18.3, 3.1 and 20.7°, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form Bof Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has anXRPD pattern substantially the same as the X-ray powder diffractionpattern shown in Figure K, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at 2-theta=15.20 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at 2-theta=6.10 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least two specific peaks at 2-theta=15.20 and 6.10plus or minus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at 2-theta=15.2, 6.1, 16.8, 12.2, 26.1,28.4, 18.3, 3.1 and 20.7° wherein said values may be plus or minus 0.2°2-theta, measured using CuKα radiation.

In a further aspect of the invention the co-crystal of Compound A with6-hydroxy-2-naphthoic acid is in a crystalline form, Form C.

According to the invention there is provided a crystalline form, Form C,of Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at about 2-theta=8.20measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at about 2-theta=24.8°,measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least two specific peaks at about 2-theta=8.2 and24.80 measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form B,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at about 2-theta=8.2, 24.8, 18.9, 29.0,14.8, 15.5 and 16.3°, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form Cof Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has anXRPD pattern substantially the same as the X-ray powder diffractionpattern shown in Figure M.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at 2-theta=8.20 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least one specific peak at 2-theta=24.80 plus orminus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with at least two specific peaks at 2-theta=8.2 and 24.80plus or minus 0.2° 2-theta, measured using CuKα radiation.

According to the invention there is provided a crystalline form, Form C,of Compound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal which has aXRPD pattern with specific peaks at 2-theta=8.2, 24.8, 18.9, 29.0, 14.8,15.5 and 16.3° wherein said values may be plus or minus 0.2° 2-theta,measured using CuKα radiation.

When it is stated that the invention relates to a crystalline form ofCompound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal the degree ofcrystallinity is conveniently greater than about 60%, more convenientlygreater than about 80%, preferably greater than about 90% and morepreferably greater than about 95%. Most preferably the degree ofcrystallinity is greater than about 98%.

It will be understood that the 2-theta values of the X-ray powderdiffraction pattern may vary slightly from one machine to another orfrom one sample to another, and so the values quoted are not to beconstrued as absolute.

It is known that an X-ray powder diffraction pattern may be obtainedwhich has one or more measurement errors depending on measurementconditions (such as equipment or machine used). In particular, it isgenerally known that intensities in an X-ray powder diffraction patternmay fluctuate depending on measurement conditions. Therefore it shouldbe understood that Compound A, Form A, of the invention is not limitedto the crystals that provide X-ray powder diffraction patterns identicalto the X-ray powder diffraction pattern shown in Figure A, and anycrystals providing X-ray powder diffraction patterns substantially thesame as those shown in Figure A fall within the scope of the invention.Similarly, it will be understood that the Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal Form A, of the invention isnot limited to the crystals that provide X-ray powder diffractionpatterns identical to the X-ray powder diffraction pattern shown inFigures C or I, and any crystals providing X-ray powder diffractionpatterns substantially the same as those shown in Figures C or I fallwithin the scope of the invention. Similarly, it will be understood thatCompound A: 6-hydroxy-2-naphthoic acid (1:1) co-crystal Forms B and C,of the invention are not limited to the crystals that provide X-raypowder diffraction patterns identical to the X-ray powder diffractionpattern shown in Figures K and M respectively, and any crystalsproviding X-ray powder diffraction patterns substantially the same asthose shown in Figures K and M fall within the scope of the invention. Aperson skilled in the art of X-ray powder diffraction is able to judgethe substantial identity of X-ray powder diffraction patterns.

Persons skilled in the art of X-ray powder diffraction will understandthat the relative intensity of peaks can be affected by, for example,grains above 30 microns in size and non-unitary aspect ratios, which mayaffect analysis of samples. The skilled person will also understand thatthe position of reflections can be affected by the precise height atwhich the sample sits in the diffractometer and the zero calibration ofthe diffractometer.

The surface planarity of the sample may also have a small effect. Hencethe diffraction pattern data presented are not to be taken as absolutevalues. (Jenkins, R & Snyder, R. L. ‘Introduction to X-Ray PowderDiffractometry’ John Wiley & Sons 1996; Bunn, C. W. (1948), ChemicalCrystallography, Clarendon Press, London; Klug, H. P. & Alexander, L. E.(1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.2° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the X-ray powder diffraction pattern in Figures A, C, I, Kand M and when reading Tables A to E (see Example 1). Furthermore, itshould be understood that intensities might fluctuate depending onexperimental conditions and sample preparation (preferred orientation).

The compounds of Formula (I) include one or more chiral centres. To theextent a structure or chemical name in this specification does notindicate chirality, the structure or name is intended to encompass anysingle stereoisomer (i.e. any single chiral isomer) corresponding tothat structure or name, as well as any mixture of stereoisomers (e.g. aracemate). It is well-known in the art how such optically-active formscan be prepared. For example, a single stereoisomer can be obtained byisolating it from a mixtures of isomers (e.g. a racemate) using, forexample, chiral chromatographic separation. In other embodiments, asingle stereoisomer is obtained through direct synthesis from, forexample, a chiral starting material.

A particular enantiomer or diastereoisomer of a compound describedherein may be more active than other enantiomers or diastereoisomers ofthe same compound.

According to a further aspect of the invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,which is a single enantiomer being in enantiomer excess (% ee) of ≧95%,≧98%, or ≧99%. Conveniently a single enantiomer is present in anenantiomer excess of ≧99%.

According to a further aspect of the invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,which is a single enantiomer being in enantiomer excess (% ee) in therange 95 to 100%.

According to a further aspect of the invention, there is provided apharmaceutical composition, which comprises a compound of Formula (I)which is a single enantiomer being in enantiomer excess (% ee) of ≧95%,≧98%, or ≧99% or a pharmaceutically acceptable salt thereof, inassociation with a pharmaceutically-acceptable diluent or carrier.Conveniently, the single enantiomer is present in an enantiomer excessof ≧99%.

According to a further aspect of the invention, there is provided apharmaceutical composition, which comprises a compound of Formula (I)which is a single enantiomer being in enantiomer excess (% ee) in therange 95 to 100%, or a pharmaceutically acceptable salt thereof, inassociation with a pharmaceutically-acceptable diluent or carrier.

The invention is intended to include all isotopes of atoms occurring inthe present compounds. Isotopes will be understood to include thoseatoms having the same atomic number but different mass number. Forexample, isotopes of hydrogen include tritium and deuterium and isotopesof carbon include ¹³C and ¹⁴C.

The term “pharmaceutically acceptable” is used to specify that an object(for example a salt, dosage form, diluent or carrier) is suitable foruse in patients. An example list of pharmaceutically acceptable saltscan be found in the Handbook of Pharmaceutical Salts: Properties,Selection and Use, P. H. Stahl and C. G. Wermuth, editors,Weinheim/Zirich:Wiley-VCH/VHCA, 2002. A suitable pharmaceuticallyacceptable salt of a compound of Formula (I) is, for example, anacid-addition salt. An acid addition salt of a compound of Formula (I)may be formed by bringing the compound into contact with a suitableinorganic or organic acid under conditions known to the skilled person.An acid addition salt may for example be formed using an inorganic acidselected from the group consisting of hydrochloric acid, hydrobromicacid, sulphuric acid and phosphoric acid. An acid addition salt may alsobe formed using an organic acid selected from the group consisting oftrifluoroacetic acid, citric acid, maleic acid, oxalic acid, aceticacid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaricacid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonicacid and para-toluenesulfonic acid.

It will be understood that the compounds of Formula (I), andpharmaceutically acceptable salts thereof, may exist in solvated andunsolvated forms. For example, a solvated form may be a hydrated form.It is to be understood that the invention encompasses all such solvatedand unsolvated forms.

The compounds of Formula (I) maybe administered in the form of aprodrug, which is a compound which that is broken down in the human oranimal body to release the compound of the invention. Such,pharmaceutically acceptable, prodrugs of compounds for Formula (I) alsoform an aspect of the invention. Various forms of prodrugs are known inthe art. For example, see

-   a) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);-   b) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and    Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991);-   c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);-   d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285    (1988); and-   e) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984).

Another aspect of the invention provides a process for preparing acompound of Formula (I), or a pharmaceutically acceptable salt thereof.A suitable process is illustrated by the following representativeprocess in which, unless otherwise stated, R¹, R² and n have themeanings defined hereinbefore. Necessary starting materials may beobtained by standard procedures of organic chemistry. The preparation ofsuch starting materials is described in conjunction with the followingrepresentative process variants and within the accompanying Examples.Alternatively, necessary starting materials are obtainable by analogousprocedures to those illustrated and are within the ordinary skill of anorganic chemist.

Compounds of Formula (I) are conveniently made by a coupling reaction,for example, reaction of a compound of Formula (II) with a compound ofFormula (IIIa) or Formula (IIIb) in the presence of a trialkylphosphine, such as trialkyl tributylphosphine, and a diazene reagent,such as (E)-diazene-1,2-diylbis(piperidin-1-ylmethanone, in a suitablesolvent, such as dichloromethane, and a suitable temperature, such as 5°C.

Compounds of Formula (II) may be made by, for example, reaction of acompound of Formula (IV) with 4-(piperidin-4-yl)phenol in the presenceof a base, such as N,N-diisopropylethylamine, in a suitable solvent,such as ethanol, and a suitable temperature, such as 55° C.

Compounds of Formula (IV) may be made by, for example, reaction of3-chloro-6-hydrazinylpyridazine with a tetramethoxyalkane, such astetramethoxymethane, at a suitable temperature such as 90° C.

Compounds of Formula (IIIa) can be made by reacting1,3-dimethylpiperazin-2-one hydrochloride with 2-bromoethanol with abase, such as potassium carbonate, in a solvent, such as2-methyltetrahydrofuran, at a suitable temperature, such as 100° C.

Compounds of Formula (IIIb) can be made by reacting1-(3,5-dimethylpiperazin-1-yl)ethanone (compound V) with2-bromopropan-1-ol with a base, such as potassium carbonate, in asolvent, such as 2-methyltetrahydrofuran, at a suitable temperature,such as 80° C.

1-(3,5-dimethylpiperazin-1-yl)ethanone can be made by reactingN-acetyl-N-(2-(trifluoromethyl)phenyl)acetamide with2,6-dimethylpiperazine in a solvent, such as ethanol, at a suitabletemperature, such as ambient temperature.

N-acetyl-N-(2-(trifluoromethyl)phenyl)acetamide can be made by reactingacetyl chloride with 2-(trifluoromethyl)aniline and pyridine in asuitable solvent such as toluene at a suitable temperature, such as 50°C.

4-(Piperidin-4-yl)phenol can be made, for example, according to thefollowing reaction scheme (Scheme 1)

In Scheme (1), the following reaction conditions can be used:—

step (a): a base, such as lithium bis(trimethylsilyl)amide and asulfonylating agent, such as1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide,in the presence of a solvent, such as THF, at a suitable temperature,such as between −78 to 0° C.;step (b): 4-hydroxyphenylboronic acid in the presence of a palladium IIcatalyst, such as1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II), a base, suchas sodium carbonate and a solvent, such as dioxane-water, at a suitabletemperature, such as 80° C.; andstep (c) hydrogen in the presence of a hydrogenation catalyst, such as5% palladium on carbon, in a solvent, such as methanol.

Compounds of Formula (I) may also be made by, for example, by reactionof a compound of Formula (VIa) or a compound of Formula (VIb) with acompound of Formula (IV), as described above, in the presence of a base,such as triethylamine, in a suitable solvent, such as dimethylformamide, and at a suitable temperature, such as 56° C.

Compounds of Formula (VIa) can be made by reacting compounds of -Formula(VIIa) with an acid, such as hydrogen chloride, in the presence of asuitable solvent, such as dioxane, and a suitable temperature such 20°C.

Compounds of Formula (VIIa) can be made by reacting compounds of Formula(VIIIa) with 1,3-dimethylpiperazine-2-one in the presence of a base,such as N,N-diisopropylethylamine, in the presence of a catalyst, suchas potassium iodide, and a solvent, such as dimethylacetamide, and asuitable temperature, such as 120° C.

Compounds of Formula (VIIa) can be made by reacting tert-butyl4-(4-hydroxphenyl)piperidine-1-carboxylate with 1-bromo-3-chloroalkaneand a base, such as potassium carbonate, and a solvent, such asdichloromethane, and at a suitable temperature, such as 80° C.

Tert-butyl 4-(4-hydroxphenyl)piperidine-1-carboxylate can be made byreacting 4-(piperidin-4-yl)phenol (made as hereinbefore described) withdi-tert-butyl dicarbonate in a suitable solvent, such asdichloromethane, and a suitable temperature, such as 0° C.

Compounds of Formula (VIb) can be made by reacting compounds of Formula(VIIb) in the presence of a suitable solvent, such as methanol, and asuitable catalyst, such as 10% palladium on carbon, under an atmosphereof hydrogen.

Compounds of Formula (VIIb) can be made by reacting compounds of Formula(VIIIb) with 1-(3,5-dimethylpiperazin-1-yl)ethanone, made as describedabove, in the presence of a suitable base, such asN,N-diisopropylethylamine, in the presence of a catalyst, such aspotassium iodide, and a solvent, such as dimethylacetamide, and at asuitable temperature, such as 120° C.

Compounds of Formula (VIIIb) can be made by reacting benzyl4-(4-hydroxphenyl)piperidine-1-carboxylate with a 1-bromo-3-chloroalkaneand a base, such as potassium carbonate, and a solvent, such asdichloromethane, and at a suitable temperature, such as 80° C.

Benzyl 4-(4-hydroxphenyl)piperidine-1-carboxylate can be made byreacting 4-(piperidin-4-yl)phenol (made as hereinbefore described) withbenzylchloroformate and DIPEA in a suitable solvent, such asdichloromethane, and a suitable temperature.

As stated above, one aspect of invention is a co-crystal of Compound Awith 6-hydroxy-2-naphthoic acid.

The co-crystal can be prepared by mixing Compound A in a suitablesolvent with 6-hydroxy-2-naphthoic acid in a suitable solvent. Thus,according to a further aspect of the invention, there is provided amethod of preparing a co-crystal of Compound A with6-hydroxy-2-naphthoic acid, the method comprising the step of mixing asolution of Compound A which is in suitable solvent with6-hydroxy-2-naphthoic acid which is in a suitable solvent. Suitablesolvents would include solvents that solubilise both components and donot form solvates with either Compound A or 6-hydroxy-2-naphthoic acid.According to a further aspect of the invention there is provided aCompound A: 6-hydroxy-2-naphthoic acid co-crystal obtainable by thesteps of

-   -   i) mixing a solution of Compound A in suitable solvent with        6-hydroxy-2-naphthoic acid in a suitable solvent; and    -   ii) drying the resultant mixture from step (i) to obtain a        solid.

In one aspect of the invention the suitable solvent is methanol.

Compound A:6-hydroxy-2-naphthoic acid (1:1) co-crystal was found to havea number of advantageous properties compared to the free base form ofCompound A. In particular, it was found to be significantly lesshydroscopic than Compound A free base. The co-crystal was also found tobe more stable than Compound A free base when exposed to range oftemperature and humidity conditions.

Biological Assays—

The following assays were used to measure the effects of the compoundsof the present invention.

BROMOscan™ Assay (Ex Discoverx)

The ability of the compounds to bind to a bromodomain protein was testedby Discoverx using their proprietary ligand binding site-directedcompetition assay. Supplied compounds were anonymised.

The BROMOscan assay is based on the principle that test compounds thatbind the bromodomain protein prevent its binding to an immobilizedligand thus reducing the amount of protein captured on a solid support.Conversely, test molecules that do not bind the bromodomain have noeffect on the amount of protein captured on the solid support. Screening“hits” are identified by measuring the amount of bromodomain captured intest versus control samples by using a quantitative, precise andultra-sensitive qPCR method that detects the associated DNA label. In asimilar manner, dissociation constants (Kds) for testcompound-bromodomain interactions are calculated by measuring the amountof bromodomain protein captured on the solid support as a function ofthe test compound concentration.

Alpha-Screen Assay

The ability of the compounds to bind to a bromodomain protein was testedin an AlphaScreen® assay. The assay is based on the interaction betweenHistidine-tagged bromodomain protein which can bind to Nickel-chelatedonor beads, and a Biotinylated acetyl lysine peptide corresponding to ahistone amino acid sequence, which can bind to streptavidin-conjugatedacceptor beads. The protein-peptide interaction can be detected by lightemission at 520-620 nm. In the presence of compounds which bind to BRD4a lower signal is observed as the protein-peptide interaction isreduced.

-   -   1. The assay was performed as follows:—Greiner BioOne (cat        no. 784075) compound plates were used. Compounds were prepared        using the Labcyte Echo Acoustic Dispenser with compounds in a        final volume of 40 nl per well normalised to 0.5% (v/v) DMSO        under final assay conditions. Compounds were tested in 12 point        singlicate concentration response format.    -   2. 4 μl of BRD4 protein (6His-TEV-BRD4, amino acid residues        42-169, corresponding to the BD1 domain) (final assay        concentration=50 nM) per well was added using the Beckman        Coulter BioRAPTR® Flying Reagent Dispensor Microfluidic        Workstation.    -   3. Incubated for 30 minutes at room temperature.    -   4. Added 4 μl of acetyl lysine peptide        (H4K5,8,12,16(Ac)₄-biotin:(NH2-)        YSGRG(K-Ac)GG(K-Ac)GLG(K-Ac)GGA(K-Ac)RHR(K-Biotin)(-COOH))        (final assay concentration=50 nM) per well using the Beckman        Coulter BioRAPTR® Flying Reagent Dispensor Microfluidic        Workstation.    -   5. Incubated for 30 minutes at room temperature.    -   6. Added 4 μl of Nickel & Streptavidin-bead solution pre-mixed        (beads supplied by Perkin Elmer) per well using the BioRaptr as        before (final assay concentration=4 μg/ml). Kept plates in the        dark after addition.    -   7. Incubated for 60 minutes at room temperature keeping the        assay plate in the dark    -   8. Plates were then read using the Perkin Elmer Envision plate        reader, laser excitation at 680 nm and emission detected at        520-620 nm.    -   9. Data was analysed using Genedata software and IC₅₀ values        calculated.

Anti-Proliferative Assay

The anti-proliferative effect of the compounds was assessed byAlamarBlue assay in MM1.S cells which were originally derived from amultiple myeloma patient. This assay is based on Resazurin, anon-fluorescent indicator dye converted to bright red-fluorescentresorufin via the reduction reactions of metabolically active cells. Theamount of fluorescence produced is proportional to the number of livingcells. MM.1S cells are cultured in RPMI-1640 medium (Gibco®) plus 10%Fetal bovine serum (FBS) and 1 mM L-glutamine. 12-24 hours beforecompound dosing, 90 μL of cell suspension (18,750 cells) was seeded into96-well microtiter plates (black, flat bottom). On the day of compounddosing, compounds were serially diluted 1:3 in 100% DMSO using columns2-10 of a 96-well microtiter plate. Column 11 of compound serial platecontained only DMSO. All wells were then further diluted 1:30 in media.10 μL of compound or DMSO alone in media was added to cell platescolumns 2-11 in triplicate. In addition, 1 plate had 10 μL of mediaadded and was developed using alamar blue. Plates developed on the dayof compound addition were referred to as Day 0. Dosed plates werecultured 3 days under normal conditions (RPMI-1640 plus 10% FBS and 1 mML-glutamine) After 3 days of culture, dosed plates are developed usingeither MTS or alamar blue. For each compound concentration, % net growthwas calculated by (Day 3 dosed well-Average Day 0)/(Average Day 3 DMSOcontrol-Average Day 0). The GI₅₀, the concentration that causes 50% ofgrowth inhibition, of each compound was calculated using the % netgrowth as defined by National Cancer Institute (NCI).

Assay Monitoring cMyc Protein Modulation

Multiple myeloma MM1.S cells were cultured in RPMI-1640 mediumcontaining 10% FBS and 1% L-glutamine under standardized conditions in ahumidified incubator (37° C. and 5% CO₂). The impact of cMyc proteinmodulation induced by bromodomain inhibitors was assessed by stainingand quantifying c-Myc protein level after compound treatment using aflow cytometer Assay, carried out in 96-well plate format with 200Kcells per well. Cells were treated with serially diluted compounds 16hours before fixing with 2% paraformaldehyde (final concentration) for10 minutes at 37° C. After being permeabilized by ice cold 90% methanolat 4° C. for 30 min, cells were washed, blocked by buffer (0.5% FBS inphosphate-buffered saline (PBS) buffer) for 10 minutes at roomtemperature, and stained with cMyc antibody for 1 hour (Cell SignalingTechnology®5605, 1:200 dilution). Cells were washed and stained byincubating with Alexa-488 conjugated anti-Rabbit IgG (Cell SignalingTechnology®#4412, 1:1000 dilution) at RT for 30 minutes. After staining,cells were washed again and fixed with 2% paraformaldehyde in PBS andready for analysis by BD FACSCalibur™ flow cytometer. Fluorescencegeometric mean was calculated through FlowJo (TreeStar Inc), maximalinhibition signal was determined by control compound treatment at highdose across each plate, and minimal inhibition signal was determined byDMSO treatment. IC50 was calculated by fitting the dose-response datapoints which are normalized against max and min signals as percentage ofinhibition using a standard 4-Parameter-Logistic nonlinear regressionmodel.

Although the pharmacological properties of the compounds of the Formula(I) vary with structural change as expected, in general, activitypossessed by compounds of the Formula (I) may be demonstrated at thefollowing concentrations or doses in one or more of the above tests.

The following data was generated for the Examples (the data below may bea result from a single experiment or an average of multiple repeatexperiments):

TABLE X Alpha screen DiscoverX K_(D)/μM BRD4 (1) BRD4 BRD4 BRD2 BRD2BRD3 BRD3 BRDT BRDT Example IC₅₀/μM (1) (2) (1) (2) (1) (2) (1) (2) 10.12 0.047 0.13 0.10 0.28 0.064 0.43 0.095 0.45 (n = 2) (n = 2) (n = 4)(n = 2) (n = 2) (n = 2) (n = 2) (n = 2) (n = 2) 2 0.035 0.026 0.29 0.0830.35 0.057 0.92 0.035 1.1 (n = 2) (n = 6) (n = 6) (n = 2) (n = 2) (n =2) (n = 2) (n = 2) (n = 2) 3 0.30 0.11 0.34 0.16 0.53 0.11 0.67 0.0930.99 (n = 2) (n = 2) (n = 4) (n = 2) (n = 2) (n = 2) (n = 2) (n = 2) (n= 2) 4 0.14 0.057 0.25 (n = 4) (n = 2) (n = 2)Number of repeats in parentheses

TABLE Y MM1.S Antiproliferative Example GI₅₀/μM MM1.S “MoA” IC₅₀/μM 10.0049 (n = 9)  0.011 (n = 12) 2 <0.0020 (n = 3)  <0.0020 (n = 4)  30.0075 (n = 2) 0.025 (n = 1) 4 0.0051 (n = 3) 0.012 (n = 2)Number of repeats in parentheses

Xenograft Models

Compound A was also investigated in a xenograft model as describedbelow.

Female CB17 SCID mice aged 6 to 8 weeks were obtained from Charles RiverLaboratories (Wilmington, Mass.) and maintained underspecific-pathogen-free conditions in an AAALAC (Association forAssessment and Accreditation of Laboratory Animal Care)-accreditedfacility. Irradiated food and autoclaved water were provided ad libitum.

MV-4-11 cells (American Tissue Culture Consortium) were resuspended in0.1 ml of medium without serum and Matrigel (Becton Dickinson) at a 1:1ratio. Cells (10⁷/mouse) were injected subcutaneously into the rightflank of mice. Tumors were allowed to grow until they reached an averagevolume of 200 mm³ for efficacy and then the mice were randomized intogroups of 8. Compound A was solubilized in 0.5% HPMC/0.1% Tween80 fordosing. For efficacy, either vehicle or Compound A was administered POonce a day (qd) for 21 days at 10 mg/kg (mpk). Body weight and tumourvolume were measured twice a week for 21 days. The results are shown inFigure E and demonstrate the effect of Compound A on tumour volume.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of Formula (I),or a pharmaceutically acceptable salt thereof, as defined herein, inassociation with a pharmaceutically acceptable diluent or carrier.According to a further aspect of the invention, there is provided apharmaceutical composition, which comprises a CompoundA:6-hydroxy-2-naphthoic acid co-crystal, as defined herein, inassociation with a pharmaceutically acceptable diluent or carrier.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, hard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder) or for parenteral administration (for example as a sterileaqueous or oily solution for intravenous, subcutaneous, intramuscular orintramuscular dosing or as a suppository for rectal dosing). In oneaspect of the invention the pharmaceutical composition of the inventionis a composition suitable for oral use.

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

For further information on formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of editorial Board), Pergamon Press 1990.

The compound of Formula (I) or the Compound A:6-hydroxy-2-naphthoic acidco-crystal will normally be administered to a warm-blooded animal at aunit dose within the range of 5 to 5000 mg/m² body area of the animali.e. approximately 0.1 to 100 mg/kg, and this normally provides atherapeutically effective dose. A unit dose form, such as a tablet or acapsule, will usually contain 0.5 mg to 250 mg and, such as, 1 to 250 mgof active ingredient. The daily dose will necessarily be varieddepending upon the animal or patient host treated, the particular routeof administration, and the severity of the illness being treated.Accordingly, the practitioner who is treating any particular animal orpatient may determine the optimum dosage. The compounds or co-crystalsof the present invention are potentially of value as anti-proliferativeagents and/or cell killing agents in the containment and/or treatment ofhaematological cancers (also referred to as liquid cancers) and solidtumour disease. Particularly, the compounds or co-crystals of theinvention are expected to be useful in the prevention or treatment ofthose tumours which are associated with amplification of one or more ofthe BET family of bromodomain containing proteins, suitably BRD4amplification, or are dependent on key oncogenes which can be regulatedby one or more of the BET family of bromodomain containing proteins,suitably BRD4, for example, ovarian, acute myeloid and mixed lineageleukemia (AML), multiple myeloma (MM), diffuse large B-cell lymphoma(DLBCL), castration-resistant prostate cancer (CRPC), non-small celllung cancer (NSCLC), small cell lung cancer (SCLC), breast cancer,glioblastoma, and neuroblastoma.

The term “therapy” is intended to have its normal meaning of dealingwith a disease in order to entirely or partially relieve one, some orall of its symptoms, or to correct or compensate for the underlyingpathology. The term “therapy” also includes “prophylaxis” unless thereare specific indications to the contrary. The terms “therapeutic” and“therapeutically” should be interpreted in a corresponding manner.

The term “prophylaxis” is intended to have its normal meaning andincludes primary prophylaxis to prevent the development of the diseaseand secondary prophylaxis whereby the disease has already developed andthe patient is temporarily or permanently protected against exacerbationor worsening of the disease or the development of new symptomsassociated with the disease.

The term “treatment” is used synonymously with “therapy”. Similarly theterm “treat” can be regarded as “applying therapy” where “therapy” is asdefined herein.

The term ‘effective amount’ refers to the amount of a compound ofFormula (I) or co-crystal as described in any of the embodiments hereinwhich is effective to potentially provide therapy in a subject. In thecase of cancer, the effective amount may cause any of the changesobservable or measurable in a subject as described in the definition of“therapy”, “treatment” and “prophylaxis” For example, the effectiveamount can potentially reduce the number of cancer or tumour cells;potentially reduce the overall tumour size; potentially inhibit or stoptumour cell infiltration into peripheral organs including, for example,the soft tissue and bone; potentially inhibit and stop tumourmetastasis; potentially inhibit and stop tumour growth; potentiallyrelieve to some extent one or more of the symptoms associated with thecancer; potentially reduce morbidity and mortality; potentially improvequality of life; or a combination of such effects. An effective amountmay be an amount sufficient to decrease the symptoms of a diseaseresponsive to inhibition of one or more bromodomain-containing proteins.For cancer therapy, efficacy in-vivo can, for example, be measured byassessing the duration of survival, time to disease progression (TTP),the response rates (RR), duration of response, and/or quality of life.

According to the present invention, there is provided a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, or acompound of Formula (I):co-former co-crystal, suitably a CompoundA:6-hydroxy-2-naphthoic acid co-crystal, as defined hereinbefore, foruse in therapy.

According to a further aspect of the invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for the manufacture of a medicament.

According to the present invention, there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof, or a CompoundA:6-hydroxy-2-naphthoic acid co-crystal, as defined hereinbefore, foruse as a medicament in a warm-blooded animal such as man.

According to a further aspect of the invention, there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in the production of anti-proliferative orcell-killing effect in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use in theproduction of an anti-proliferative or cell-killing effect in awarm-blooded animal such as man.

According to a further aspect of the invention, there is provided theuse of a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, for the production of an anti-proliferative orcell-killing effect in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided amethod for producing an anti-proliferative or cell-killing effect in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof Formula (I), or a pharmaceutically acceptable salt thereof, or aCompound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in the prevention or treatment of cancer in a warmblooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use in theprevention or treatment of cancer in a warm-blooded animal such as man.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of cancer in a warm-bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, or aCompound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in the prevention or treatment of haematologicalcancers (also referred to a liquid cancers) and solid cancers in a warmblooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use in theprevention or treatment of haematological and solid cancers in awarm-blooded animal such as man.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of haematological and solidcancers in a warm-blooded animal, such as man, in need of such treatmentwhich comprises administering to said animal an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

One aspect of the invention provides compounds of Formula (I) thatpotentially inhibit one or more bromodomain-containing proteins, i.e.the BET family of bromodomain-containing proteins, and such as BRD2,BRD3, BRD4, and BRDt and, suitably BRD4. Advantageously such compoundsmay be useful for the treatment of a proliferative disorder such ascancer in a patient where the proliferative disorder is abromodomain-containing protein mediated disorder. By‘bromodomain-containing protein mediated disorder’ is meant any diseaseor other deleterious condition in which one or more of thebromodomain-containing proteins are known to play a role.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in the prevention or treatment of a BET dependentcancer in a warm blooded animal such as man.

By BET dependent cancer we mean any cancer in which one or more of theBET family of bromodomain-containing proteins such as BRD2, BRD3, BRD4,and BRDt play a role.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in the prevention or treatment of a BRD4 dependentcancer in a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use in theprevention or treatment of a BET dependent cancer in a warm-bloodedanimal such as man.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use in theprevention or treatment of a BRD4 dependent cancer in a warm-bloodedanimal such as man.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of BET dependent cancer in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof Formula (I), or a pharmaceutically acceptable salt thereof, or aCompound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

According to a further aspect of the invention there is provided amethod for the prevention or treatment of BRD4 dependent cancer in awarm-blooded animal, such as man, in need of such treatment whichcomprises administering to said animal an effective amount of a compoundof Formula (I), or a pharmaceutically acceptable salt thereof, or aCompound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in providing an inhibitory effect on one or moreof the BET family of bromodomain-containing proteins.

According to a further aspect of the invention there is provided acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore, for use in providing an inhibitory effect on BRD4.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use inproviding an inhibitory effect on one or more of the BET family ofbromodomain-containing proteins.

According to a further aspect of the invention there is provided the useof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic acid co-crystal, asdefined hereinbefore, in the manufacture of a medicament for use inproviding an inhibitory effect on BRD4.

According to a further aspect of the invention there is provided amethod for providing an inhibitory effect on one or more of the BETfamily of bromodomain-containing proteins which comprises administeringto said animal an effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof, or a CompoundA:6-hydroxy-2-naphthoic acid co-crystal, as defined hereinbefore.

According to a further aspect of the invention there is provided amethod for providing an inhibitory effect on BRD4 which comprisesadministering to said animal an effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt thereof, or aCompound A:6-hydroxy-2-naphthoic acid co-crystal, as definedhereinbefore.

The Compound A:6-hydroxy-2-naphthoic acid co-crystal can be in Form A, Bor C, as herein defined and suitably is in Form A.

The anti-cancer treatment described herein may be applied as a soletherapy or may involve, in addition to the compounds of the invention,conventional surgery or radiotherapy or chemotherapy or immunotherapy.Such chemotherapy could be administered concurrently, simultaneously,sequentially or separately to treatment with the compound of theinvention.

Where a combination therapy is used, the amount of the compound ofFormula (I) or pharmaceutically acceptable salt thereof or CompoundA:6-hydroxy-2-naphthoic acid co-crystal described in this specificationand the amount of the other pharmaceutically active agent(s) are, whencombined, jointly effective to treat a targeted disorder in the animalpatient. In this context, the combined amounts are in a “therapeuticallyeffective amount” if they are, when combined, sufficient to decrease thesymptoms of a disease responsive to inhibition of one or morebromodomain-containing proteins. Typically, such amounts may bedetermined by one skilled in the art by, for example, starting with thedosage range described in this specification for the compound of Formula(I) or pharmaceutically acceptable salt thereof or a CompoundA:6-hydroxy-2-naphthoic acid co-crystal as herein defined and anapproved or otherwise published dosage range(s) of the otherpharmaceutically active compound(s).

In a further aspect of the invention there is provided a pharmaceuticalproduct comprising the compound of Formula (I), and an additionalanti-tumour substance, for the conjoint treatment of cancer.

In such an aspect of the invention there is provided a pharmaceuticalproduct comprising the compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore, and an additionalanti-tumour agent, for the conjoint treatment of cancer.

In such aspects the pharmaceutical product comprises a compound ofFormula (I) and co-former in form of a co-crystal.

In a further aspect of the invention there is provided a pharmaceuticalproduct comprising a Compound A:6 hydroxy-2-naphthoic acid co-crystal,as defined hereinbefore, and an additional anti-tumour substance, forthe conjoint treatment of cancer.

Such conjoint treatment may involve one or more of the followinganti-tumour agent:—

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil, pemetrexel and tegafur,raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea);antitumour antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); and topoisomerase inhibitors (for exampleepipodophyllotoxins like etoposide and teniposide, amsacrine, topotecanand camptothecin and irinotecan); inhibitors of DNA repair mechanismssuch as CHK kinase; DNA-dependent protein kinase inhibitors; inhibitorsof poly (ADP-ribose) polymerase (PARP inhibitors, including olaparib);and Hsp90 inhibitors such as tanespimycin and retaspimycin;

(ii) compounds that inhibit progression through the cell cycle such asantimitotic agents (for example vinca alkaloids like vincristine,vinblastine, vindesine and vinorelbine; epothilones such as ixabepiloneand patupilone; taxoids like taxol, taxotere and docetaxel; polo-likekinase inhibitors; and inhibitors of kinesin motor proteins such as Eg5protein inhibitors); aurora kinase inhibitors (for example AZD1152,PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459);cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;and inhibitors of centromeric protein function such as CENP-Einhibitors;

(iii) cytostatic agents that alter hormone-dependent growth such asantioestrogens (for example tamoxifen, fulvestrant, toremifene,raloxifene, droloxifene and iodoxyfene), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane); inhibitors of 5α-reductase such as finasteride and CYP17A1inhibitors such as abiraterone;

(iv) anti-invasion agents, for example c-Src kinase family inhibitorslike AZD0530 (saracatinib); dasatinib ((BMS-354825), J. Med. Chem.,2004, 47, 6658-6661) and bosutinib (SKI-606), and metalloproteinaseinhibitors like marimastat, inhibitors of urokinase plasminogenactivator receptor function or antibodies to heparanase; inhibitors ofFAK or focal-adhesion kinase; small molecule inhibitors of MET receptorkinase (for example volitinib/AZD6904); antibodies to MET receptorkinase or the MET ligand hepatocyte growth factor (for exampleonartuzumab);

(v) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such as gefitinib (ZD1839), erlotinib (OSI-774) and CI 1033,erbB2 tyrosine kinase inhibitors such as lapatinib; mixed erbB 1/2inhibitors such as afatanib; and irreversible inhibitors of EGFR andHer2 such as HKI-272, irreversible inhibitors of EGFR such as AZD9291;inhibitors of the hepatocyte growth factor family and their receptors;inhibitors of the insulin growth factor family including small moleculekinase inhibitors and antibodies directed to insulin-like growth factorsand insulin-like growth factor receptors; inhibitors of theplatelet-derived growth factor family and their receptors such asimatinib and/or nilotinib (AMN107); c-kit inhibitors, AnLK inhibitors,Flt3 kinase inhibitors, c-abl kinase inhibitors, and inhibitors ofCSF-1R or TRK kinase;

(vi) inhibitors of signal transduction kinases as FGFR (for exampleAZD4547), PIM (for example AZD1208), MEK (for example Selumetinib(AZD6244), AKT (for example AZD5363), inhibitors of TOR kinases(including TORC1 and TORC2, for example AZD2014), and inhibitors of PI3kinase, including isoforms such as PI3K-α, PI3K-β or PI3K-δ (for exampleAZD8186); inhibitors of serine/threonine kinases such as Ras or Rafkinases (for example sorafenib or vemurafenib); Inhibitors of PDK, SGK,P14K or PIP5K, JAK, STAT (including STAT3, an inhibitor of which isAZD9150) and IRAK4; ATR inhibitors (for example AZD6738) or ATMinhibitors, BTK inhibitors such as ibrutinib, SYK inhibitors such asfostamatinib, and cyclin dependent kinase inhibitors; farnesyltransferase inhibitors such as tipifarnib (R115777) lonafarnib(SCH66336) and Wee-li kinase inhibitors (for example AZD1775 asdescribed in WO2007/126128);

(vii) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™) and forexample, a VEGF receptor tyrosine kinase inhibitor such as vandetanib(ZD6474), sorafenib, vatalanib (PTK787), sunitinib (SU11248), axitinib(AG-013736), pazopanib (GW 786034) and cediranib (AZD2171), compoundssuch as those disclosed in WO97/22596, WO97/30035, WO97/32856 andWO98/13354 and compounds that work by other mechanisms (for examplelinomide, inhibitors of integrin αvβ3 function and angiostatin);

(viii) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in WO99/02166, WO00/40529, WO00/41669, WO01/92224, WO02/04434and WO02/08213;

(ix) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;

(x) gene therapy approaches, including for example approaches to replaceaberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT(gene-directed enzyme pro-drug therapy) approaches such as those usingcytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy;

(xi) immunotherapy approaches, including for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor; approaches to decreaseT-cell anergy or regulatory T-cell function; approaches that enhanceT-cell responses to tumours, such as blocking antibodies to CTLA4 (forexample ipilimumab and tremelimumab), B7H1, PD-1 (for example BMS-936558or MEDI-4736), and agonist antibodies to CD137; approaches usingtransfected immune cells such as cytokine-transfected dendritic cells;approaches using cytokine-transfected tumour cell lines, approachesusing antibodies to tumour associated antigens, and antibodies thatdeplete target cell types (e.g., unconjugated anti-CD20 antibodies suchas Rituximab, radiolabeled anti-CD20 antibodies Bexxar and Zevalin, andanti-CD54 antibody Campath); R-CHOP chemotherapy regimen (Rituximabtogether with cyclophosphamide, doxorubicin hydrochloride, vincristinesulphate and prednisone); approaches using anti-idiotypic antibodies;approaches that enhance Natural Killer cell function; and approachesthat utilize antibody-toxin conjugates (e.g. anti-CD33 antibodyMylotarg); immunotoxins such as moxetumumab pasudotox; agonists oftoll-like receptor 7 or toll-like receptor 9;

(xii) inhibitors of proteasome mediated protein degradation includingbut not limited to proteasome inhibitors such as Velcade™ (Bortezomib)or carfilzomib, inhibitors of ubiquity lipases, inhibitors of ubiquitinproteases, inhibitors of protein Neddylation, and inhibitors of proteinsumoylation; and

(xiii) other standard of care agents such as cyclophosphamide,prednisone, lenalidomide or thalidomide.

According to this aspect of the invention there is provided acombination suitable for use in the treatment of cancer comprising acompound of Formula (I) or a pharmaceutically acceptable salt thereofand an additional anti-tumour agent, in particular any one of theanti-tumour agents listed in (i)-(xiii) above.

According to this aspect of the invention there is also a provided acombination suitable for use in the treatment of cancer comprising aCompound A:6-hydroxy-2-naphthoic acid co-crystal and an additionalanti-tumour agent, in particular any one of the anti-tumour agentslisted in (i)-(xiii) above.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an additional anti-tumour agent, in particular an anti-tumour agentselected from one listed in (i)-(xiii) above.

In a further aspect of the invention there is provided a compound A:6-hydroxy-2-naphthoic co-crystal in combination with an additionalanti-tumour agent, in particular an anti-tumour agent selected from onelisted in (i)-(xiii) above.

According to a further aspect of the invention there is provided a kitcomprising a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, or a Compound A:6-hydroxy-2-naphthoic co-crystal incombination with an anti-tumor agent. In certain embodiments, the kitadditionally comprises instructions for the use of said compound(s) orco-crystal.

According to a further aspect of the invention there is provided a kitcomprising:

a) a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a Compound A:6-hydroxy-2-naphthoic co-crystal in a firstunit dosage form;

b) an additional anti-tumor agent in a second unit dosage form; and

c) container means for containing said first and second dosage forms.

The invention will now be illustrated in the following examples inwhich, generally:

(i) temperatures are given in degrees Celsius (° C.); unless statedotherwise, operations were carried out at room or ambient temperature,that is, at a temperature in the range of 18 to 25° C.;(ii) organic solutions were dried over anhydrous magnesium sulfate oranhydrous sodium sulfate; evaporation of solvent was carried out using arotary evaporator under reduced pressure (600 to 4000 Pascals; 4.5 to 30mmHg) with a bath temperature of up to 60° C.;(iii) chromatography means flash chromatography on silica gel; thinlayer chromatography (TLC) was carried out on silica gel plates;(iv) in general, the course of reactions was followed by TLC and/oranalytical LC-MS, and reaction times where given are for illustrationonly;(v) final products had satisfactory proton nuclear magnetic resonance(NMR) spectra and/or mass spectral data;(vi) yields are given for illustration only and are not necessarilythose which can be obtained by diligent process development;preparations were repeated if more material was required;(vii) when given, NMR data is in the form of delta values for majordiagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard, determined at 300, 400or 500 MHz using perdeuterio dimethyl sulfoxide (DMSO-d₆) as solventunless otherwise indicated; the following abbreviations have been used:s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broadsinglet; dd, double doublet; td, triple doublet; qd, quartet doublet;(viii) For the carbon (¹³C) cross polarisation magic angle spinningsolid state NMR analysis carried out on Example 1, spectra of theco-crystal, free base of Compound A and co-former were recorded on aBruker Avance NMR spectrometer operating at a ¹H frequency of 400 MHz.The samples were spun about the magic angle at a frequency of 9 kHz anda contact pulse of 2 ms was used to allow transfer of magnetisation fromproton to carbon. A recycle delay of 5 s was used to allow for spinlattice relaxation;(ix) For the nitrogen (¹⁵N) cross polarisation magic angle spinningsolid state NMR analysis carried out on Example 1, spectra of theco-crystal were recorded on a Bruker Avance NMR spectrometer operatingat a ¹H frequency of 400 MHz. The samples were spun about the magicangle at a frequency of 5 kHz and a contact pulse of 200 μs and 2 ms wasused to allow transfer of magnetisation from proton to carbon. A recycledelay of 5 s was used to allow for spin lattice relaxation;(x) chemical symbols have their usual meanings; SI units and symbols areused;(xi) Mass spectra (MS) and LC-MS data were generated on an LC-MS systemwhere the HPLC component comprised generally either an Agilent 1100,Waters Alliance HT (2790 & 2795) equipment or an HP1100 pump and DiodeArray with CTC autosampler and was run on a Phenomenex Gemini C18 5 μm,50×2 mm column (or similar) eluting with either acidic eluent (forexample, using a gradient between 0-95% water/acetonitrile with 5% of a1% formic acid in 50:50 water:acetonitrile (v/v) mixture), or basiceluent (for example, using a gradient between 0-95% water/acetonitrilewith 5% of a 0.1% 880 ammonia in acetonitrile mixture); and the MScomponent comprised generally a Waters ZQ mass spectrometer scanningover an appropriate mass range. Chromatograms for Electrospray (ESI)positive and negative Base Peak Intensity, and UV Total AbsorptionChromatogram from 220-300 nm, are generated and values for m/z aregiven; generally, only ions which indicate the parent mass are reportedand unless otherwise stated the value quoted is the (M+H)+ for positiveion mode and (M−H)− for negative ion mode;(xii) unless stated otherwise compounds containing an asymmetricallysubstituted carbon have not been resolved;(xiii) preparative high performance liquid chromatography (HPLC) wasperformed on a Gilson instrument using the following conditions:—Column: C18 reversed-phase silica, for example, Waters ‘Xbridge’, 5 μmsilica, 19×100 mm, or 30×100 mm, using decreasingly polar solventmixtures as eluent (decreasing ratio of solvent A to solvent B); solventA: water with 1% ammonium hydroxide; solvent B: acetonitrile; flow rate:28 ml/min or 61 ml/min; gradient: tailored to suit eachcompound—generally 7-10 min in length; wavelength: 254 nm;(xiv) Strong cation exchange (SCX) chromatography was performed onpre-packed cartridges (for example, ISOLUTE SCX-2 propyl sulfonicacid-based cartridges supplied by International Sorbent Technology),using a basic eluent (for example, 1M ammonia in methanol);(xv) the following abbreviations have been used herein, wherenecessary:—ADDP 1,1′-(azodicarbonyl)dipiperidineDCM dichloromethane

DIPEA N,N-diisopropylethylamine

DMA N,N-dimethyl acetamide

DMF N,N-dimethylformamide DME Dimethoxyethane

DMSO dimethylsulphoxideEt₂O diethyletherEtOAc ethyl acetateEtOH ethanolHPLC high performance liquid chromatographyMeOH methanolMgSO₄ magnesium sulfateMTBE methyl tert-butyl etherNMR nuclear magnetic resonanceSCX strong cation exchangeTFA trifluoroacetic acidTHF tetrahydrofuran;(xvii) For XRPD analysis of Example 1, the sample was mounted on asilicon wafer mount and analysed using the PANalytical CubiX PROdiffractometer. Samples were measured in reflection geometry in θ-2θconfiguration over the scan range 2° to 40° 2θ with a nominal 25 secondexposure per 0.02° increment. The sample was spun at 30 revolutions perminute (to improve counting statistics) and irradiated with X-raysgenerated by a copper long-fine focus tube operated at 45 kV and 40 mAwith a wavelength of 1.5418 Å. Persons skilled in the art of X-raypowder diffraction will understand that the relative intensity of peakscan be affected by, for example, grains above 30 microns in size andnon-unitary aspect ratios that may affect analysis of samples. Theskilled person will also understand that the position of reflections canbe affected by the precise height at which the sample sits in thediffractometer and the zero calibration of the diffractometer. Thesurface planarity of the sample may also have a small effect. Hence thediffraction pattern data presented are not to be taken as absolutevalues.(xviii) Differential Scanning Calorimetry: Analytical Instrument: TAInstruments Q1000 DSC.

Typically less than 5 mg of material contained in a standard aluminiumpan fitted with a lid was heated over the temperature range 25° C. to300° C. at a constant heating rate of 10° C. per minute. A purge gasusing nitrogen was used—flow rate 50 ml per minute.

EXAMPLE 1: PREPARATION OF(R)-4-(2-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)ETHYL)-1,3-DIMETHYLPIPERAZIN-2-ONEFORM A

Tributylphosphine (102 mL, 414.92 mmol) was added portionwise to asuspension of4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol(67.5 g, 207.46 mmol) in well degassed, anhydrous DCM (1.7 L) at 5° C.under nitrogen. The mixture was cooled to 0° C. and(E)-diazene-1,2-diylbis(piperidin-1-yl methanone) (105 g, 414.92 mmol)was added portionwise. A solution of(R)-4-(2-hydroxyethyl)-1,3-dimethylpiperazin-2-one (46.4 g, 269.70 mmol)in DCM (200 mL) was then added dropwise. The reaction mixture wasstirred for 30 minutes and filtered. The clear solution was diluted withfurther DCM (1 L) and then acidified with 2M HCl (400 mL) and water (400mL) was added. The combined aqueous solution was washed with DCM (3×1 L)and then EtOAc (1 L). The aqueous solution was then basified with solidNa₂CO₃ to pH-10 and extracted with DCM (3×1.5 L). The combined organicsolution was washed with water (500 mL) and saturated brine (500 mL),then dried over MgSO₄ and evaporated to dryness to afford crudematerial. This was purified by flash silica chromatography, eluting withEtOH:EtOAc:heptane:NH_(3(aq)) 1.8:4:4:0.2. Fractions containing thedesired product were evaporated to dryness to give a yellow foam. Thiswas further purified by preparative HPLC (Chiralpak AS column, 20 msilica, 100 mm diameter, 250 mm length), heptane/EtOH 50/50 at 400ml/min. Fractions containing the desired product were evaporated todryness and the resulting solid was stirred as a suspension in diethylether (300 mL) for 18 hours, filtered and dried to afford(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one(69 g, 69.4%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO, 30° C.)1.22 (3H, d), 1.62 (2H, qd), 1.82 (2H, d), 2.6-2.79 (3H, m), 2.79 (3H,s), 2.85-3.09 (4H, m), 3.13 (1H, q), 3.2-3.26 (2H, m), 4.03 (2H, t),4.17 (3H, s), 4.28 (2H, d), 6.85 (2H, d), 7.15 (2H, d), 7.29 (1H, d),7.85 (1H, d). m/z ES+ [M+H]+ 480

The4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenolused as starting material was prepared as follows:—

Preparation of benzyl4-(trifluoromethylsulfonyloxy)-5,6-dihydropyridine-1(2H)-carboxylate

A solution of benzyl 4-oxopiperidine-1-carboxylate (88.57 g, 379.70mmol) in THF (300 mL) was added dropwise to lithiumbis(trimethylsilyl)amide (1M in THF) (418 mL, 417.67 mmol) at −78° C.,over a period of 1 hour under nitrogen. The resulting mixture wasstirred at −78° C. for 90 minutes then a solution of1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide(142 g, 398.68 mmol) in THF (600 mL) was added dropwise over a period of1 hour. The resulting mixture was stirred at −78° C. for 30 minutes,then allowed to warm to ambient temperature and stirred for 16 hours.The reaction mixture was quenched with 2M aqueous sodium hydroxide (450mL). The layers were separated and the organic layer was washed with 2Maqueous sodium hydroxide (360 mL). The solvent was evaporated, then theresidue was re-dissolved in Et₂O (1500 mL) and the solution washed withwater (500 mL). The organic layer was dried over MgSO₄, filtered andevaporated to afford benzyl4-(trifluoromethylsulfonyloxy)-5,6-dihydropyridine-1(2H)-carboxylate(124 g, 81%) as a colourless oil. ¹H NMR (400 MHz, DMSO, 30° C.) 2.43(2H, m), 3.62 (2H, m), 4.06 (2H, m), 5.10 (2H, s), 6.02 (1H, m), 7.34(5H, m).

Preparation of benzyl 4-(4-hydroxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate

Sodium carbonate (96 g, 909.79 mmol) was added to benzyl4-(trifluoromethyl sulfonyloxy)-5,6-dihydropyridine-1(2H)-carboxylate(123.1 g, 303.26 mmol) and 4-hydroxyphenylboronic acid (46.0 g, 333.59mmol) in a mixture of dioxane (1000 mL) and water (250 mL). Theresulting mixture was bubbled with nitrogen for 10 minutes then1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (5.49 g, 7.58mmol) was added and the reaction mixture was heated at 80° C. for 1hour. The reaction mixture was diluted with DCM (2 L) and washed withwater (2 L). The aqueous layer was re-extracted with DCM (1 L), then thecombined organics were washed with saturated brine (500 mL), dried overMgSO₄, filtered and evaporated to afford crude product. The crudeproduct was purified by flash silica chromatography, elution gradient 10to 30% EtOAc in isohexane. Fractions containing the desired product wereevaporated to dryness then triturated with isohexane, filtered and driedto afford benzyl4-(4-hydroxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (62.3 g,66.4%) as a white solid. ¹H NMR (400 MHz, DMSO, 30° C.) 2.44 (2H, m),3.61 (2H, m), 4.05 (2H, m), 5.12 (2H, s), 5.99 (1H, m), 6.73 (2H, d),7.26 (2H, d), 7.32-7.40 (5H, m), 9.45 (1H, s). m/z: ES+ [M+H]+ 310.

Preparation of 4-(piperidin-4-yl)phenol

Benzyl 4-(4-hydroxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (37.7g, 121.86 mmol) and 5% palladium on carbon (7.6 g, 3.57 mmol) in MeOH(380 mL) were stirred under an atmosphere of hydrogen at 5 bar and 25°C. for 2 hours. The catalyst was removed by filtration, washed with MeOHand the solvents evaporated. The crude material was triturated with Et₂O(200 mL), then the desired product collected by filtration and driedunder vacuum to afford 4-(piperidin-4-yl)phenol (20.36 g, 94%) as awhite solid. ¹H NMR (400 MHz, DMSO, 30° C.) 1.46 (2H, m), 1.65 (2H, m),2.45 (1H, m), 2.58 (2H, m), 3.02 (2H, m), 6.68 (2H, d), 7.00 (2H, d),9.15 (1H, s). m/z: ES+ [M+H]+ 178

Preparation of 4-(piperidin-4-yl)phenol hydrobromide

Hydrogen bromide (48% in water) (0.283 mL, 2.48 mmol) was added dropwiseto a suspension 4-(piperidin-4-yl)phenol (0.4 g, 2.26 mmol) in THF (23mL). The resulting suspension was stirred for 30 minutes. The solid wascollected by filtration, washed with THF (20 mL) and dried under vacuumto give 4-(piperidin-4-yl)phenol hydrobromide (0.580 g, 100%) as a whitepowder. 1H NMR (400 MHz, DMSO, 30° C.) 1.74 (2H, qd), 1.86 (2H, d), 2.71(1H, tt), 2.96 (2H, td), 3.33 (2H, d), 6.68-6.73 (2H, m), 6.97-7.02 (2H,m), 8.48 (2H, br s), 9.18 (1H, br s).

Preparation of 6-chloro-3-methoxy-[1,2,4]triazolo[4,3-b]pyridazine

3-Chloro-6-hydrazinylpyridazine (18 g, 124.51 mmol) was suspended in DME(330 mL) and treated with tetramethoxymethane (26.5 mL, 199.22 mmol) andthe resulting mixture was stirred at 90° C. for 3 hours. The DME wasevaporated off and the residue was dissolved in 5% MeOH/DCM; and thenfiltered through a silica plug. The filtrate was evaporated to drynessand then taken up in MTBE (200 mL) and slurried for 1 hour. The solidwas filtered and dried under vacuum to afford6-chloro-3-methoxy-[1,2,4]triazolo[4,3-b]pyridazine (19.78 g, 86%) as acream powder. ¹H NMR (400 MHz, DMSO, 30° C.) 4.25 (3H, s), 7.30 (1H, d),8.22 (1H, d). m/z: ES+ [M+H]+ 185

Preparation of4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol

6-Chloro-3-methoxy-[1,2,4]triazolo[4,3-b]pyridazine (19.73 g, 106.91mmol) was added to 4-(piperidin-4-yl)phenol hydrobromide (18.4 g, 71.28mmol) in EtOH (200 mL). To this mixture was added DIPEA (62.2 mL, 356.38mmol) and the reaction was stirred at 55° C. for 18 hours. The reactionmixture was then cooled to ambient temperature and poured intovigorously stirred water (1600 mL); and stirred vigorously for 2 hours.The solid precipitate was filtered off and washed sequentially with H₂O(200 mL) and Et₂O (200 mL). The resulting solid was dried under vacuumto afford4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol(15.30 g, 66.0%) as a pale brown solid. ¹H NMR (400 MHz, DMSO, 30° C.)1.59 (2H, qd), 1.81 (2H, d), 2.67 (1H, ddt), 2.9-3.02 (2H, m), 4.17 (3H,s), 4.23-4.31 (2H, m), 6.63-6.71 (2H, m), 7.02 (2H, dd), 7.29 (1H, d),7.84 (1H, d), 9.14 (1H, s). m/z ES+ [M+H]+ 326

The (R)-4-(2-hydroxyethyl)-1,3-dimethylpiperazin-2-one used as startingmaterial was prepared as follows:—

Preparation of (R)-4-(2-hydroxyethyl)-1,3-dimethylpiperazin-2-one

2-Bromoethanol (108 mL, 1518.54 mmol) was added to a mixture of(R)-1,3-dimethylpiperazin-2-one hydrochloride (50 g, 303.71 mmol) andpotassium carbonate (126 g, 911.12 mmol) in 2-methyltetrahydrofuran (500mL). The mixture was stirred at 100° C. for 16 hours. The mixture wasfiltered and evaporated to dryness to give crude product. This waspurified by flash chromatography on silica gel eluting with 1 to 5% MeOHin DCM and pure fractions were combined and evaporated to dryness toafford (R)-4-(2-hydroxyethyl)-1,3-dimethylpiperazin-2-one (36.0 g,68.8%) as a thick yellow oil. 5 ¹H NMR (400 MHz, DMSO, 30° C.) 1.19 (3H,d), 2.42 (1H, dt), 2.59 (2H, tt), 2.79 (3H, s), 2.93-3.1 (2H, m),3.17-3.25 (2H, m), 3.47 (2H, q), 4.41 (1H, t).

The final product,(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one,was analysed by XRPD and DSC and found to be crystalline. XRPD of asample of the material gave rise to a diffraction pattern as shown inFigure A.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-oneForm A is characterised by at least one peak at a 2θ value of 20.9° or16.7°, measured using CuKα radiation. The ten most prominent peaks ofthe XRPD are shown in Table A.

TABLE A Ten most Prominent XRPD peaks for (R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one Form A Angle 2- Theta (2θ) Intensity % 20.9100.0 16.7 53.4 20.2 38.1 21.2 27.2 27.4 26.5 18.0 23.4 16.8 20.0 23.618.1 15.1 14.2 15.5 13.9wherein the 2-theta values are +/−0.2°.

Differential Scanning calorimetry (DSC) analysis of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-oneForm A showed a melting endotherm with an onset of 106.4° C. and a peakat 111.2° C. A trace of the DSC is shown in Figure B.

EXAMPLE 1.1: PREPARATION OF(R)-4-(2-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)ETHYL)-1,3-DIMETHYLPIPERAZIN-2-ONE:6-HYDROXY-2-NAPHTHOICACID (1:1) CO-CRYSTAL, FORM A

Approximately 3 g of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-oneForm A was added to a round bottom flask containing 10 mL of methanol. Aseparate solution containing 1 molar equivalent (1.18 g) of6-hydroxy-2-naphthoic acid in 5 mL methanol was then added dropwise tothe round bottom flask, and the reaction was stirred overnight at roomtemperature. The material was filtered the following day and washed withmethanol (5 mL). The recovered solid was air dried and then transferredto a vacuum oven where it was further dried overnight at 50° C.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal was obtained as an off whitesolid. This form was determined to be crystalline by XRPD.

This material was analysed by XRPD and DSC. XRPD of a sample of thematerial gave rise to a diffraction pattern as shown in Figure C.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form A is characterised by at least one peak at a2θ value of 19.5° or 12.5°, measured using CuKα radiation. The ten mostprominent peaks of the XRPD are shown in Table B.

TABLE B Ten most Prominent XRPD peaks for (R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal FormA Angle 2- Theta (2θ) Intensity % 19.5 100 12.5 80.4 18.1 79.8 12.8 66.424.2 60.9 14.1 56.5 23.4 51.8 17.9 40.2 18.6 38.6 17.0 37.3

wherein the 2-theta values are +/−0.2°.

Differential Scanning calorimetry (DSC) analysis of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form A showed a melting endotherm with an onset of186.3° C. and a peak at 188.3° C. A trace of the DSC is shown in FigureD.

Co-crystals can be defined in terms of the ΔpKa, i.e.(pKa(base)−pKa(acid)). If ΔpKa is <1, the API:coformer molecule complexis classified as a co-crystal. (Regulatory Classification ofPharmaceutical Co-Crystals, US FDA Guidance, April 2013). The pKa forthe basic centre on the piperazinone in Compound A was determined to be4.8 and the pKa for the co-former molecule 6-hydroxy-2-naphthoic acid4.3, which gives a ΔpKa is <1 and, therefore is consistent with theformation of a co-crystal.

¹³C cross polarisation magic angle spinning solid state NMR analysis wascarried out on the final product of Example 1.1,(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-oneand the 6-hydroxy-2-naphthoic acid co-former. The spectra are shown inFigure F. The bottom spectra in Figure F (i.e. of the product of Example1.1) was not sum of the spectra of the(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one(middle spectra) and the co-former (top spectra). In the top spectrathere was a peak at about 172 ppm, attributed to the fully protonatedcarboxylic acid in the co-former. (If the carboxylic acid in theco-former is not protonated, then a peak would be expected at 177 ppmrather than 172 ppm). In the middle spectra there was a peak at about169 ppm attributed to the carbonyl in(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one.In the spectra for the product of Example 1.1, there were 3 peaks in thecarbonyl region which are not consistent with the peaks in the top ormiddle spectra. Furthermore, in this spectra, there was no peak at 177ppm. Such a peak would be expected to be present if the co-formercarboxylic acid is not protonated and would be indicative that a protonhad transferred between the co-former and free base and a salt formed.The absence of this peak is consistent with formation of a co-crystal.

¹⁵N cross polarisation magic angle spinning solid state NMR analysis wascarried out on the final product of Example 1.1. Spectra were recordedat contact times of 2 ms and 200 μs and are shown in Figure G. Thespectrum recorded with the longer contact time was consistent with atleast 8 different nitrogen environments in the co-crystal whereas at theshorter contact time no peaks are observed. This was consistent withnone of the nitrogen atoms exhibiting a strong dipolar coupling to aproton as would be the case if a proton had fully transferred betweenthe conformer and the base as would be observed for a salt.

The stoichiometry of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid co-crystal was determined by proton NMR. The material gave rise toa NMR spectrum as shown in Figure H. Stoichiometry was determined byintegration of resonance due to(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one,for instance using the resonance at 6.85 ppm (2H) and comparison to aresonance due to 6-hydroxy-2-naphthoic acid, for instance using theresonance at 8.46 (1H), and determining the ratio between the peaks,allowing for the number of protons giving rise to the resonance signal.Stoichiometry (molar ratio) was determined to be 1:1.

¹H NMR (500 MHz, DMSO, 27° C.) 1.22 (3H, d), 1.62 (2H, qd), 1.82 (2H,d), 2.63-2.79 (3H, m), 2.81 (3H, s), 2.85-3.09 (4H, m), 3.13 (1H, q),3.20-3.28 (2H, m), 4.03 (2H, t), 4.17 (3H, s), 4.28 (2H, d), 6.85 (2H,d), 7.12-7.21 (4H, m), 7.29 (1H, d), 7.75 (1H, d), 7.83-7.89 (2H, m),7.96 (1H, d), 8.47 (1H, s), 10.15 (1H, bs), 12.81 (1H, bs)

Thus, the ¹H NMR, ¹³C and ¹⁵N solid state NMR and the ΔpKa, referred toabove, were consistent with the formation of CompoundA:6-hydroxy-2-naphthoic acid (1:1) co-crystal

EXAMPLE 1.2 PREPARATION OF(R)-4-(2-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)ETHYL)-1,3-DIMETHYLPIPERAZIN-2-ONE:6-HYDROXY-2-NAPHTHOICACID (1:1) CO-CRYSTAL FORM A

4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol(0.818 kg, 2.34 mol) was mixed with ADDP (1.19 kg, 4.67 mol) and DCM(9.8 L, 150 mol) and stirred at about 10° C. Tributyl phosphine (0.98kg, 47.6 mol) was added portionwise to the reaction mixture over 30minutes and then it was stirred for 30 minutes. A solution of(R)-4-(2-hydroxyethyl)-1,3-dimethylpiperazin-2-one (0.503 kg, 2.80 mol)in DCM (1.64 L, 25.6 mol was then added dropwise and the reactionmixture was stirred for 24 hours.

The reaction mixture was then filtered by washing with DCM to remove theADDP by-product. The filtrate was stirred with aqueous hydrochloric acidand the lower organic layer discarded. The aqueous layer was furtherwashed with DCM and the lower organic layer discarded. The aqueoussolution was then basified with Na₂CO₃ to pH 9-10 and extracted withDCM. The DCM layer was further washed with water and evaporated andazeotroped with methanol to remove residual water to afford crudematerial. The crude material was dissolved in methanol (7.5 L, 190 mol)and heated to 60° C. in vessel 1. 6-hydroxynaphthalene-2-carboxylic acid(0.360 kg, 1.87 mol) was dissolved in methanol (3.8 L, 94 mol) in vessel2. 10% of the solution from vessel 2 was then added to vessel 1 dropwiseover 10 minutes. The temperature of vessel 1 was maintained atapproximately 60° C.

Compound A:6-hydroxy-2-napthoic acid (1:1) co-crystal seed material (1.2g, 0.0018 mol), which can be made as described in Example 1.1, was addedto vessel 1 and the temperature held at 60° C. for approximately 1 hour.The remaining contents of vessel 2 were then added to vessel 1 dropwiseover approximately 16 hours. The resultant slurry was cooled to roomtemperature over 5 hours and then filtered and washed with methanol. Therecovered solid was dried in a vacuum oven at 50° C. to afford(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal (56.45% yield).

¹H NMR (500 MHz, DMSO-d6) ä ppm 1.43 (d, J=7.00 Hz, 3H) 1.54-1.69 (m,2H) 1.80 (d, J=11.36 Hz, 2H) 2.74 (tt, J=12.06, 3.41 Hz, 1H) 2.84 (s,3H) 2.91-3.03 (m, 2H) 3.25-3.63 (m, 6H) 3.83 (d, J=6.88 Hz, 1H)4.10-4.34 (m, 7H) 6.89 (d, J=8.69 Hz, 2H) 7.09-7.22 (m, 4H) 7.28 (d,J=10.34 Hz, 1H) 7.72 (d, J=8.72 Hz, 1H) 7.79-7.88 (m, 2H) 7.92 (d,J=8.88 Hz, 1H) 8.44 (d, J=0.63 Hz, 1H) 10.12 (br. S., 1H). m/z (ES+),[M+H]+=480.

This form was determined to be crystalline by XRPD.

Preparation of4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol

3-Chloro-6-hydrazinylpyridazine (0.753 kg) was mixed withtetramethoxymethane (8.231 mol, 1.22 kg) in methanol (5.7 L) andstirred. The resulting mixture was then heated and stirred at 55° C. for2 hours. After cooling to 45° C., 4-(piperidin-4-yl)phenol hydrobromide(prepared as described above) (1.000 kg, 3.874 mol) was added. DIPEA(2.03 L, 11.6 mol) was then added dropwise over a period of about 10minutes and the reaction was further stirred. Methanol (5.1 L, 126 mol)was added and the reaction mixture was stirred for at least 48 hours atapproximately 45° C. The mixture was filtered and the filtrate waswashed with methanol and water. The isolated solid was dried in a vacuumoven at approximately 50° C. to afford4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol(65% yield).

¹H NMR (400 MHz, DMSO, 30° C.) 1.59 (2H, qd), 1.81 (2H, d), 2.67 (1H,ddt), 2.9-3.02 (2H, m), 4.17 (3H, s), 4.23-4.31 (2H, m), 6.63-6.71 (2H,m), 7.02 (2H, dd), 7.29 (1H, d), 7.84 (1H, d), 9.14 (1H, s). m/z ES+[M+H]+ 326

(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal was analysed by XRPD and DSC. XRPD of a sample ofthe material gave rise to the diffraction pattern shown in Figure I.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form A is characterised by at least one peak at a2θ value of 19.4° or 12.5°, measured using CuKα radiation. The ten mostprominent peaks of the XRPD are shown in Table C.

TABLE C Ten most Prominent XRPD peaks for (R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal FormA Angle 2- Theta (2θ) Intensity % 19.4 100 12.5 79.3 12.8 77.4 18.1 75.024.2 66.8 23.4 55.2 14.0 53.2 18.6 37.8 17.0 37.5 17.9 36.4

wherein the 2-theta values are +/−0.2°.

Differential Scanning calorimetry (DSC) analysis of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form A showed a melting endotherm with an onset of184.9° C. and a peak at 187.9° C. (Figure J).

Thus DSC analysis showed of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal Form A is a high meltingsolid with an onset of melting in the range of 163186° C. and a peak inthe range of 169-188° C.

EXAMPLE 1.1A—MATERIAL MADE IN A REPEAT PREPARATION OF THE ROUTEDESCRIBED IN EXAMPLE 1.1, RESULTED IN A FURTHER FORM, FORM B

This form was determined to be crystalline by XRPD.

XRPD of a sample of the material gave rise to a diffraction pattern asshown in Figure K.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form B is characterised by at least one peak at a2θ value of 15.2° or 6.1°, measured using CuKα radiation. The nine mostprominent peaks of the XRPD are shown in Table D.

TABLE D Nine most Prominent XRPD peaks for (R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal FormB Angle 2- Theta (2θ) Intensity % 15.2 40.9 6.1 58.1 16.8 64.3 12.2 44.026.1 43.9 28.4 41.0 18.3 34.2 3.1 30.6 20.7 25.4

wherein the 2-theta values are +/−0.2°.

Differential Scanning calorimetry (DSC) analysis of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form B showed a melting endotherm with an onset of169.3° C. and a peak at 172.7° C. A trace of the DSC is shown in FigureL.

EXAMPLE 1.3: PREPARATION OF(R)-4-(2-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)ETHYL)-1,3-DIMETHYLPIPERAZIN-2-ONE:6-HYDROXY-2-NAPHTHOICACID (1:1) CO-CRYSTAL, FORM C

A sample of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form A was analysed by hot stage XRPD using aBruker D8 advance diffractometer. The sample was heated to 210° C. withdiffractograms collected every 3° C.

The sample was then cooled to 25° C. at 10 C/min, and upon opening thesample stage at the end of the experiment material was observed to havesublimed and collected on the beam knife of the diffractometer as awhite powder. This white powder was collected and analysed and shown tobe a different crystal form, Form C. This form was determined to becrystalline by XRPD.

XRPD of a sample of the material gave rise to a diffraction pattern asshown in Figure M.(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form C is characterised by at least one peak at a2θ value of 8.2° or 24.8°, measured using CuKα radiation. The seven mostprominent peaks of the XRPD are shown in Table E.

TABLE E Seven most Prominent XRPD peaks for (R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoic acid (1:1) co-crystal FormC Angle 2- Theta (2θ) Intensity % 8.2 100 24.8 90.9 18.9 46.4 29.0 32.314.8 26.3 15.5 22.2 16.3 20.7

wherein the 2-theta values are +/−0.2°.

Differential Scanning calorimetry (DSC) analysis of(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one:6-hydroxy-2-naphthoicacid (1:1) co-crystal Form C showed a melting endotherm with an onset of156.8° C. and a peak at 160.5° C. A trace of the DSC is shown in FigureN.

EXAMPLE 2: PREPARATION OF1-((3S,5R)-4-(2-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)ETHYL)-3,5-DIMETHYLPIPERAZIN-1-YL)ETHANONE

DIPEA (1.455 mL, 8.36 mmol) was added to1-((3S,5R)-3,5-dimethyl-4-(2-(4-(piperidin-4-yl)phenoxy)ethyl)piperazin-1-yl)ethanone(1.502 g, 4.18 mmol) and6-chloro-3-methoxy-[1,2,4]triazolo[4,3-b]pyridazine (obtained asdescribed in Example 1, preparation of starting materials) (1.003 g,5.43 mmol) in DMF (15 mL). The resulting solution was stirred at 80° C.for 18 hours and evaporated to dryness. The crude product was purifiedby ion exchange chromatography, using an SCX column. The desired productwas eluted from the column using 1M NH₃/MeOH and evaporated to drynessto afford a brown gum. This was further purified by flash silicachromatography, elution gradient 0 to 10% 7M NH₃/MeOH in EtOAc. Purefractions were evaporated to dryness to afford1-((3S,5R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-3,5-dimethylpiperazin-1-yl)ethanone(0.991 g, 46.7%) as a cream foam.

¹H NMR (400 MHz, DMSO, 100° C.) 1.06-1.1 (6H, m), 1.69 (2H, qd), 1.91(2H, d), 1.97 (3H, s), 2.56-2.68 (4H, m), 2.78 (1H, tt), 2.99 (2H, t),3.06 (2H, td), 3.84 (2H, br s), 4.00 (2H, t), 4.21 (3H, s), 4.27 (2H,d), 6.83-6.88 (2H, m), 7.14-7.19 (3H, m), 7.74 (1H, d). m/z: ES+ [M+H]+508

The1-((3S,5R)-3,5-dimethyl-4-(2-(4-(piperidin-4-yl)phenoxy)ethyl)piperazin-1-yl)ethanoneused as starting material was prepared as follows:—

Preparation of benzyl 4-(4-hydroxyphenyl)piperidine-1-carboxylate

Benzyl chloroformate (5.97 mL, 41.84 mmol) was added to4-(piperidin-4-yl)phenol hydrobromide (obtained as described in Example1, preparation of starting materials) (9 g, 34.86 mmol) and DIPEA (14.57mL, 83.67 mmol) in DCM (150 mL). The resulting suspension was stirredfor 2 hours. The reaction mixture was washed sequentially with water(2×100 mL) and 1M aqueous citric acid (100 mL). The organic layer wasdried over MgSO₄, filtered and evaporated to afford crude product. Thecrude product was purified by flash silica chromatography, elutiongradient 0 to 5% MeOH in DCM. Pure fractions were evaporated to drynessto afford benzyl 4-(4-hydroxyphenyl)piperidine-1-carboxylate (7.89 g,72.7%) as a colourless gum, which solidified on standing. 1H NMR (400MHz, DMSO, 30° C.) 1.43 (2H, qd), 1.71 (2H, d), 2.57 (1H, tt), 2.79-2.93(2H, m), 4.11 (2H, d), 5.08 (2H, s), 6.64-6.69 (2H, m), 6.98-7.02 (2H,m), 7.28-7.33 (1H, m), 7.34-7.4 (4H, m), 9.10 (1H, s). m/z: ES+ [M+H]+312

Preparation of benzyl4-(4-(2-chloroethoxy)phenyl)piperidine-1-carboxylate

1-Bromo-2-chloroethane (2.134 mL, 25.64 mmol) was added to benzyl4-(4-hydroxyphenyl)piperidine-1-carboxylate (5.322 g, 17.09 mmol) andpotassium carbonate (4.72 g, 34.18 mmol) in MeCN (80 mL). The resultingmixture was stirred at 85° C. for 18 hours. The reaction was incompleteand further potassium carbonate (4.72 g, 34.18 mmol) and1-bromo-2-chloroethane (2.134 mL, 25.64 mmol) were added and the mixturewas stirred at 85° C. for a further 48 hours. The reaction showed someprogress to ˜50% completion. The reaction was incomplete so thetemperature was increased to 95° C. and the reaction mixture was stirredfor a further 24 hours. The reaction mixture was evaporated to drynessand redissolved in EtOAc (200 mL), and washed sequentially with water(2×100 mL) and saturated brine (100 mL). The organic layer was driedover MgSO₄, filtered and evaporated to afford crude product. The crudeproduct was purified by flash silica chromatography, elution gradient 0to 5% MeOH in DCM. Pure fractions were evaporated to dryness to affordbenzyl 4-(4-(2-chloroethoxy)phenyl)piperidine-1-carboxylate (3.30 g,51.7%) as a pale yellow gum. 1H NMR (400 MHz, DMSO, 30° C.) 1.47 (2H,qd), 1.73 (2H, d), 2.64 (1H, tt), 2.81-2.95 (2H, m), 3.90 (2H, dd), 4.12(2H, d), 4.20 (2H, dd), 5.08 (2H, s), 6.85-6.9 (2H, m), 7.12-7.17 (2H,m), 7.28-7.34 (1H, m), 7.34-7.4 (4H, m). m/z: ES+ [M+H]+ 374

Preparation of benzyl4-(4-(2-((2S,6R)-4-acetyl-2,6-dimethylpiperazin-1-yl)ethoxy)phenyl)piperidine-1-carboxylate

DIPEA (3.05 mL, 17.49 mmol) was added to benzyl4-(4-(2-chloroethoxy)phenyl)piperidine-1-carboxylate (2.18 g, 5.83mmol), 1-((3S,5R)-3,5-dimethylpiperazin-1-yl)ethanone (1.366 g, 8.75mmol) and potassium iodide (0.968 g, 5.83 mmol) in DMA (25 mL). Theresulting mixture was stirred at 125° C. for 18 hours. The reactionmixture was evaporated to dryness and redissolved in EtOAc (250 mL), andwashed sequentially with water (200 mL) and saturated brine (200 mL).The organic layer was dried over MgSO₄, filtered and evaporated toafford crude product. This was purified by flash silica chromatography,elution gradient 0 to 4% 7M NH₃/MeOH in DCM. Pure fractions wereevaporated to dryness to afford benzyl4-(4-(2-((2S,6R)-4-acetyl-2,6-dimethylpiperazin-1-yl)ethoxy)phenyl)piperidine-1-carboxylate(2.180 g, 76%) as a brown gum. ¹H NMR (400 MHz, DMSO, 100° C.) 1.06-1.1(6H, m), 1.51 (2H, qd), 1.76-1.83 (2H, m), 1.97 (3H, s), 2.57-2.72 (5H,m), 2.93 (2H, td), 2.99 (2H, t), 3.85 (2H, br s), 4.00 (2H, t), 4.14(2H, d), 5.12 (2H, s), 6.83-6.87 (2H, m), 7.1-7.15 (2H, m), 7.27-7.33(1H, m), 7.34-7.38 (4H, m). m/z: ES+ [M+H]+ 494

Preparation of1-((3S,5R)-3,5-dimethyl-4-(2-(4-(piperidin-4-yl)phenoxy)ethyl)piperazin-1-yl)ethanone

Benzyl4-(4-(2-((2S,6R)-4-acetyl-2,6-dimethylpiperazin-1-yl)ethoxy)phenyl)piperidine-1-carboxylate(2.18 g, 4.42 mmol) and 10% palladium on carbon (0.470 g, 0.44 mmol) inMeOH (45 mL) were stirred under an atmosphere of hydrogen for 5 hours.The mixture was then filtered and evaporated to dryness to give1-((3S,5R)-3,5-dimethyl-4-(2-(4-(piperidin-4-yl)phenoxy)ethyl)piperazin-1-yl)ethanone(1.502 g, 95%) as a pale yellow gum. ¹H NMR (400 MHz, DMSO, 100° C.)1.07-1.1 (6H, m), 1.48 (2H, qd), 1.70 (2H, d), 1.97 (3H, s), 2.5-2.66(7H, m), 2.99 (2H, t), 3.01-3.07 (2H, m), 3.85 (2H, br s), 4.00 (2H, t),6.82-6.86 (2H, m), 7.09-7.13 (2H, m). m/z: ES+[M+H]+ 360

The 1-((3S,5R)-3,5-dimethylpiperazin-1-yl)ethanone used as startingmaterial was prepared as follows:—

Preparation of N-acetyl-N-(2-(trifluoromethyl)phenyl)acetamide

Acetyl chloride (132 mL, 1861.91 mmol) was added dropwise over 30minutes to 2-(trifluoromethyl)aniline (100 g, 620.64 mmol) and pyridine(200 mL, 2482.55 mmol) in toluene (500 mL) cooled to 0° C. The reactionwas heated to 50° C. and stirred for 20 hours. The mixture was thencooled to ambient temperature and washed twice with 1M aqueous citricacid (250 mL). The crude product mixture was then evaporated to halfvolume and treated with heptane (500 mL). The resulting slurry wasstirred at 5° C. for 4 hours and then the precipitate was collected byfiltration, washed with heptane (500 mL) and dried under vacuum. Thisgave N-acetyl-N-(2-(trifluoromethyl)phenyl)acetamide (93 g, 59.1%) as alight brown solid. ¹H NMR (400 MHz, DMSO, 30° C.) 2.18 (6H, s),7.58-7.93 (4H, m). m/z: ES+ [M+H]+ 246

Preparation of 1-((3R,5S)-3,5-dimethylpiperazin-1-yl)ethanone

N-acetyl-N-(2-(trifluoromethyl)phenyl)acetamide (13.28 g, 52.54 mmol)was added to 2R,6S-2,6-dimethylpiperazine (5 g, 43.79 mmol) in EtOH (75mL) and the mixture was stirred at ambient temperature for 24 hours.This was then evaporated to dryness, redissolved in DCM (25 mL) andwashed with 2M aqueous HCl (25 mL). The aqueous solution was thenbasified to pH 14 with concentrated aqueous NaOH and extracted with DCM(2×25 mL). The combined organics were evaporated to dryness to give ayellow liquid. This was purified by flash silica chromatography, elutiongradient 0 to 10% 7M NH₃/MeOH in DCM. Pure fractions were evaporated todryness to afford 1-((3S,5R)-3,5-dimethylpiperazin-1-yl)ethanone (4.00g, 66.7%) as a pale tan oil. ¹H NMR (400 MHz, DMSO, 100° C.) 0.98 (6H,d), 1.78 (1H, br s), 1.96 (3H, s), 2.26 (2H, br s), 2.58-2.68 (2H, m),3.94 (2H, br s).

EXAMPLE 3: PREPARATION OF(R)-4-(3-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)PROPYL)-1,3-DIMETHYLPIPERAZIN-2-ONE

Triethylamine (0.396 mL, 2.84 mmol) was added to6-chloro-3-methoxy-[1,2,4]triazolo[4,3-b]pyridazine (obtained asdescribed in Example 1, preparation of starting materials) (350 mg, 1.90mmol) and(R)-1,3-dimethyl-4-(3-(4-(piperidin-4-yl)phenoxy)propyl)piperazin-2-one(655 mg, 1.90 mmol) in DMF (10 mL) and the mixture was heated to 56° C.for 5 hours. The crude product solution was purified by ion exchangechromatography, using an SCX column. The desired product was eluted fromthe column using 7M NH₃/MeOH and evaporated to dryness to give a browngum. This was further purified by flash silica chromatography, elutiongradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to drynessto afford(R)-4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-1,3-dimethylpiperazin-2-one(140 mg, 14.96%) as a brown foam. ¹H NMR (400 MHz, DMSO, 30° C.); 1.20(3H, d), 1.64 (2H, m), 1.86 (4H, m), 2.45 (2H, m), 2.72 (2H, m), 2.82(3H, s), 3.00 (4H, m), 3.25 (2H, m), 3.98 (2H, tr), 4.19 (3H, s), 4.30(2H, m), 6.87 (2H, dd), 7.17 (2H, dd), 7.30 (1H, d), 7.86 (1H, d). m/zES+ [M+H]+=494

The(R)-1,3-dimethyl-4-(3-(4-(piperidin-4-yl)phenoxy)propyl)piperazin-2-oneused as starting material was prepared as follows:—

Preparation of tert-butyl 4-(4-hydroxyphenyl)piperidine-1-carboxylate

Triethylamine (23.76 mL, 170.44 mmol) was added slowly to4-(piperidin-4-yl)phenol hydrobromide (obtained as described in Example1, preparation of starting materials) (40 g, 154.95 mmol) in DCM (190mL) at 0° C. The resulting mixture was stirred for 20 minutes and thendi-tert-butyl dicarbonate (35.5 g, 162.69 mmol) was added. The ice bathwas removed and the reaction was stirred at ambient temperature for 2hours. The reaction mixture was washed sequentially with water (2×200mL) and saturated brine (200 mL). The organic layer was dried overMgSO₄, filtered and evaporated to afford crude product. The solid wastaken up in MTBE (150 mL) and sonicated and slurried for 2 hours. Theresulting solid was collected by filtration, washed with heptane (200mL) and dried under vacuum to afford tert-butyl4-(4-hydroxyphenyl)piperidine-1-carboxylate (36.0 g, 84%) as a creamywhite solid product. ¹H NMR (400 MHz, DMSO, 27° C.) 1.40 (9H, s), 1.44(2H, d), 1.68 (2H, d), 2.49-2.59 (1H, m), 2.76 (2H, s), 4.03 (2H, d),6.63-6.7 (2H, m), 6.96-7.04 (2H, m), 9.13 (1H, s). m/z [ES−] M−=276

Preparation of tert-butyl4-(4-(3-chloropropoxy)phenyl)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(4-hydroxyphenyl)piperidine-1-carboxylate (9.99 g, 36.02 mmol) in MeCN(200 mL) was added 1-bromo-3-chloropropane (14.27 mL, 144.07 mmol) andpotassium carbonate (19.91 g, 144.07 mmol). The reaction was stirred at80° C. for 16 hours. The reaction mixture was diluted with water (125mL), and extracted with DCM (200 mL). The organic layer was dried overMgSO₄, filtered and evaporated to afford tert-butyl4-(4-(3-chloropropoxy)phenyl)piperidine-1-carboxylate (12.75 g, 100%) asa colourless oil. ¹H NMR (400 MHz, CDCl3, 30° C.) 1.48 (9H, s),1.54-1.65 (2H, m), 1.79 (2H, d), 2.20 (2H, d), 2.59 (1H, tt), 2.78 (2H,t), 3.56 (2H, t), 4.02-4.13 (2H, m), 4.23 (2H, d), 6.8-6.87 (2H, d),7.03-7.17 (2H, d).

Preparation of (R)-tert-butyl4-(4-(3-(2,4-dimethyl-3-oxopiperazin-1-yl)propoxy)phenyl)piperidine-1-carboxylate

DIPEA (28.2 mL, 162.13 mmol) was added to a suspension of(R)-1,3-dimethylpiperazin-2-one hydrochloride (7.12 g, 43.23 mmol),tert-butyl 4-(4-(3-chloropropoxy)phenyl)piperidine-1-carboxylate (12.75g, 36.03 mmol) and potassium iodide (5.98 g, 36.03 mmol) in DMA (100mL). The solution was heated to 120° C. for 24 hours. The reactionmixture was diluted with water (200 mL) and extracted with DCM (200 mL).The organic layer was dried over MgSO₄, filtered and evaporated toafford crude product. This was purified by flash silica chromatography,eluting with 10% MeOH in EtOAc. Pure fractions were evaporated todryness to afford (R)-tert-butyl4-(4-(3-(2,4-dimethyl-3-oxopiperazin-1-yl)propoxy)phenyl)piperidine-1-carboxylate(15.50 g, 97%) as a brown oil. ¹H NMR (400 MHz, DMSO, 30° C.) 1.19-1.22(3H, d), 1.42 (9H, s), 1.71 (2H, d), 1.8-1.9 (2H, m), 1.96 (2H, s),2.37-2.49 (1H, m), 2.60 (1H, ddt), 2.80 (5H, d), 2.93-3.05 (4H, m),3.2-3.28 (2H, m), 4.05 (2H, dd), 6.8-6.9 (2H, m), 7.12 (2H, dd). m/z ES+[M+H]+=446

Preparation of1(R)-1,3-dimethyl-4-(3-(4-(piperidin-4-yl)phenoxy)propyl)piperazin-2-one

4.0M hydrogen chloride in dioxane (34.8 mL, 139.14 mmol) was added to asuspension of (R)-tert-butyl4-(4-(3-(2,4-dimethyl-3-oxopiperazin-1-yl)propoxy)phenyl)piperidine-1-carboxylate(15.5 g, 34.78 mmol) in dioxane (20 mL). The solution was stirred to 20°C. for 2 hours. The reaction mixture was evaporated to afford crudeproduct. The crude product was purified by ion exchange chromatography,using an SCX column. The desired product was eluted from the columnusing 7M NH₃/MeOH and pure fractions were evaporated to dryness toafford(R)-1,3-dimethyl-4-(3-(4-(piperidin-4-yl)phenoxy)propyl)piperazin-2-one(10.50 g, 87%) as a brown oil. ¹H NMR (400 MHz, DMSO, 30° C.) 1.21 (3H,d), 1.73-1.93 (6H, m), 2.4-2.46 (1H, m), 2.68-2.78 (2H, m), 2.80 (3H,s), 2.92-3.04 (4H, m), 3.18 (1H, d), 3.22-3.27 (2H, m), 3.35 (2H, s),3.98 (2H, t), 6.89 (2H, d), 7.13 (2H, d), 8.79 (1H, bs). m/z ES+[M+H]+=346

EXAMPLE 4: PREPARATION OF1-((3R,5S)-4-(3-(4-(1-(3-METHOXY-[1,2,4]TRIAZOLO[4,3-B]PYRIDAZIN-6-YL)PIPERIDIN-4-YL)PHENOXY)PROPYL)-3,5-DIMETHYLPIPERAZIN-1-YL)ETHANONE

Tributylphosphine (1.441 mL, 5.84 mmol) was added dropwise to4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenol(obtained as described in Example 1, preparation of starting materials)(0.95 g, 2.92 mmol),1-((3R,5S)-4-(3-hydroxypropyl)-3,5-dimethylpiperazin-1-yl)ethanone(0.751 g, 3.50 mmol) and(E)-diazene-1,2-diylbis(piperidin-1-ylmethanone) (1.473 g, 5.84 mmol) indegassed DCM (20 mL) under nitrogen. The resulting mixture was stirredfor 90 minutes and then filtered. The crude product solution waspurified by ion exchange chromatography, using an SCX column. Thedesired product was eluted from the column using 1M NH₃/MeOH andevaporated to dryness to give a pale brown gum. This was furtherpurified by flash silica chromatography, elution gradient 0 to 10% 7MNH₃/MeOH in EtOAc. Pure fractions were evaporated to dryness to afford1-((3R,5S)-4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-3,5-dimethylpiperazin-1-yl)ethanone(0.868 g, 57.0%) as a white foam. ¹H NMR (400 MHz, DMSO, 100° C.) 1.04(6H, d), 1.69 (2H, qd), 1.76-1.84 (2H, m), 1.88-1.94 (2H, m), 1.96 (3H,s), 2.51-2.55 (2H, m), 2.56-2.7 (2H, m), 2.74-2.82 (3H, m), 3.06 (2H,td), 3.81 (2H, br s), 3.98 (2H, t), 4.21 (3H, s), 4.27 (2H, d),6.83-6.87 (2H, m), 7.13-7.18 (3H, m), 7.74 (1H, d). m/z: ES+ [M+H]+ 522

The 1-((3R,5S)-4-(3-hydroxypropyl)-3,5-dimethylpiperazin-1-yl)ethanoneused as starting material was prepared as follows:—

Preparation of1-((3R,5S)-4-(3-hydroxypropyl)-3,5-dimethylpiperazin-1-yl)ethanone

3-Bromopropan-1-ol (6.41 mL, 70.84 mmol) was added to1-((3R,5S)-3,5-dimethylpiperazin-1-yl)ethanone (obtained as described inExample 2, preparation of starting materials) (6.51 g, 41.67 mmol) andpotassium carbonate (14.40 g, 104.18 mmol) in 2-methyl tetrahydrofuran(40 mL). The resulting mixture was stirred at 80° C. for 18 hours, thenfiltered and evaporated to dryness. The crude product was purified byflash silica chromatography, elution gradient 0 to 6% 7M NH₃/MeOH inDCM. Pure fractions were evaporated to dryness to afford1-((3R,5S)-4-(3-hydroxypropyl)-3,5-dimethylpiperazin-1-yl)ethanone(0.749 g, 8.39%) as a colourless oil. ¹H NMR (400 MHz, DMSO, 30° C.)0.96-1.03 (6H, m), 1.39-1.5 (2H, m), 1.96 (3H, s), 2.19-2.28 (1H, m),2.28-2.36 (1H, m), 2.39-2.47 (1H, m), 2.67-2.77 (3H, m), 3.36 (2H, t),3.60 (1H, dt), 4.12 (1H, dt), 4.36 (1H, br s).

1. A compound of Formula (I) or a pharmaceutically acceptable saltthereof

wherein:— R¹ is the group

or the group

and

denotes the point of attachment; R² is a C₁-C₄alkyl; and n is 2 or
 3. 2.A compound as claimed in claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is the group

where

denotes the point of attachment; R² is C₁-C₄ alkyl; and n is 2 or
 3. 3.A compound as claimed in claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is the group

where

denotes the point of attachment; R² is C₁-C₄ alkyl; and n is 2 or
 3. 4.A compound as claimed in claim 1 or a pharmaceutically acceptable saltthereof, wherein R¹ is the group

where

denotes the point of attachment; R² is C₁-C₄ alkyl; and n is
 2. 5. Acompound as claimed in claim 1 or a pharmaceutically acceptable saltthereof, which compound is selected from:4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one;1-(4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-3,5-dimethylpiperazin-1-yl)ethanone;4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-1,3-dimethylpiperazin-2-one;and1-(4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-3,5-dimethylpiperazin-1-yl)ethanone.6. A compound as claimed in claim 1 or a pharmaceutically acceptablesalt thereof, which compound is selected from:(R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one;1-((3S,5R)-4-(2-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-3,5-dimethylpiperazin-1-yl)ethanone;(R)-4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-1,3-dimethylpiperazin-2-one;and1-((3R,5S)-4-(3-(4-(1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)propyl)-3,5-dimethylpiperazin-1-yl)ethanone.7. A compound as claimed in claim 6 or a pharmaceutically acceptablesalt thereof, of Formula (IA):


8. A compound as claimed in claim 7 of Formula (IA).
 9. A compound asclaimed in claim 7 in a crystalline form with an XRPD pattern withspecific peaks at about 2-theta=20.9, 16.7, 20.2, 21.2, 27.4, 18.0,16.8, 23.6, 15.1 and 15.5°, measured using CuKα radiation.
 10. Aco-crystal of the compound of Formula (IA), according to claim 7, andthe co-former molecule 6-hydroxy-2-naphthoic acid.
 11. A co-crystal ofthe compound of Formula (IA), according to claim 7, and6-hydroxy-2-naphthoic acid obtainable by the steps of i) mixing asolution of the compound of Formula (IA) in suitable solvent with6-hydroxy-2-naphthoic acid co-crystal in a suitable solvent; and ii)drying the resultant mixture from step (i) to obtain a solid. 12-14.(canceled)
 15. A method for the prevention or treatment of cancer in awarm blooded animal in need of such treatment which comprisesadministering to said animal an effective amount of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof as claimed inclaim
 1. 16. (canceled)
 17. The method according to claim 15, whereinthe cancer is ovarian cancer, acute myeloid and mixed lineage leukemia(AML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL),castration-resistant prostate cancer (CRPC), non-small cell lung cancer(NSCLC), small cell lung cancer (SCLC), breast cancer, glioblastoma, orneuroblastoma.
 18. A method for the prevention or treatment of cancer ina warm blooded animal in need of such treatment which comprisesadministering to said animal an effective amount of a co-crystal of thecompound of Formula (IA) as claimed in claim
 10. 19. The methodaccording to claim 18, wherein the cancer is ovarian cancer, acutemyeloid and mixed lineage leukemia (AML), multiple myeloma (MM), diffuselarge B-cell lymphoma (DLBCL), castration-resistant prostate cancer(CRPC), non-small cell lung cancer (NSCLC), small cell lung cancer(SCLC), breast cancer, glioblastoma, or neuroblastoma.
 20. Apharmaceutical composition comprising a compound of Formula (I) or apharmaceutically acceptable salt thereof, as claimed in claim 1, and apharmaceutically acceptable diluent or carrier.
 21. A pharmaceuticalcomposition comprising a co-crystal of the compound of Formula (IA) asclaimed in claim 10, and a pharmaceutically acceptable diluent orcarrier.